Human neuronal nicotinic acetylcholine receptor and cells transformed with same DNA and mRNA encoding an--subunit of

ABSTRACT

DNA encoding human neuronal nicotinic acetylcholine receptor alpha and beta subunits, mammalian and amphibian cells containing said DNA, methods for producing alpha and beta subunits and recombinant (i.e., isolated or substantially pure) alpha subunits (specifically  alpha 4 and  alpha 7) and beta subunits (specifically  beta 4) are provided. In addition, combinations of subunits (i.e.,  alpha 1,  alpha 2,  alpha 3,  alpha 4, and/or  alpha 7 subunits in combination with  beta 4 subunits; or  beta 2,  beta 3 and/or  beta 4 subunits in combination with  alpha 4 and/or  beta 7 subunits) are provided.

The application is a divisional of U.S. application Ser. No. 08/028,031,filed Mar. 8, 1993, now abandoned.

This invention relates to DNA encoding human neuronal nicotinicacetylcholine receptor protein subunits, as well as the proteinsthemselves. In particular, human neuronal nicotinic acetylcholinereceptor α-subunit-encoding DNA, α-subunit proteins, β-subunit-encodingDNA, β-subunit proteins, and combinations thereof are provided.

BACKGROUND OF THE INVENTION

Ligand-gated ion channels provide a means for communication betweencells of the central nervous system. These channels convert a signal(e.g., a chemical referred to as a neurotransmitter) that is released byone cell into an electrical signal that propagates along a target cellmembrane. A variety of neurotransmitters and neurotransmitter receptorsexist in the central and peripheral nervous systems. Five families ofligand-gated receptors, including the nicotinic acetylcholine receptors(NAChRs) of neuromuscular and neuronal origins, have been identified(Stroud et al. (1990) Biochemistry 29:11009-11023). There is, however,little understanding of the manner in which the variety of receptorsgenerates different responses to neurotransmitters or to othermodulating ligands in different regions of the nervous system.

The nicotinic acetylcholine receptors (NAChRs) are multisubunit proteinsof neuromuscular and neuronal origins. These receptors form ligand-gatedion channels that mediate synaptic transmission between nerve and muscleand between neurons upon interaction with the neurotransmitteracetylcholine (ACh). Since various nicotinic acetylcholine receptor(NAChR) subunits exist, a variety of NAChR compositions (i.e.,combinations of subunits) exist. The different NAChR compositionsexhibit different specificities for various ligands and are therebypharmacologically distinguishable. Thus, the nicotinic acetylcholinereceptors expressed at the vertebrate neuromuscular junction invertebrate sympathetic ganglia and in the vertebrate central nervoussystem have been distinguished on the basis of the effects of variousligands that bind to different NAChR compositions. For example, theelapid α-neurotoxins that block activation of nicotinic acetylcholinereceptors at the neuromuscular junction do not block activation of someneuronal nicotinic acetylcholine receptors that are expressed on severaldifferent neuron-derived cell lines.

Muscle NAChR is a glycoprotein composed of five subunits with thestoichiometry α₂ β(γ or ε)δ. Each of the subunits has a mass of about50-60 kilodaltons (kd) and is encoded by a different gene. The α₂ β(γ orε)δ complex forms functional receptors containing two ligand bindingsites and a ligand-gated transmembrane channel. Upon interaction with acholinergic agonist, muscle nicotinic AChRs conduct sodium ions. Theinflux of sodium ions rapidly short-circuits the normal ionic gradientmaintained across the plasma membrane, thereby depolarizing themembrane. By reducing the potential difference across the membrane, achemical signal is transduced into an electrical signal that signalsmuscle contraction at the neuromuscular junction.

Functional muscle nicotinic acetylcholine receptors have been formedwith αβδγ subunits, αβγ subunits, αβδ subunits, βδγ subunits or αδsubunits, but not with only one subunit (see e.g., Kurosaki et al.(1987) FEBS Lett. 214: 253-258; Camacho et al. (1993) J. Neuroscience13:605-613). In contrast, functional neuronal AChRs (nAChRs) can beformed from α subunits alone or combinations of α and β subunits. Thelarger α subunit is generally believed to be the ACh-binding subunit andthe lower molecular weight β subunit is generally believed to be thestructural subunit, although it has not been definitively demonstratedthat the β subunit does not have the ability to bind ACh. Each of thesubunits which participate in the formation of a functional ion channelare, to the extent they contribute to the structure of the resultingchannel, "structural" subunits, regardless of their ability (orinability) to bind ACh. Neuronal AChRs (nAChRs), which are alsoligand-gated ion channels, are expressed in ganglia of the autonomicnervous system and in the central nervous system (where they mediatesignal transmission), in post-synaptic locations (where they modulatetransmission), and in pre- and extra-synaptic locations (where they mayhave additional functions).

DNA encoding NAChRs has been isolated from several sources. Based on theinformation available from such work, it has been evident for some timethat NAChRs expressed in muscle, in autonomic ganglia, and in thecentral nervous system are functionally diverse. This functionaldiversity could be due, at least in part, to the large number ofdifferent NAChR subunits which exist. There is an incompleteunderstanding, however, of how (and which) NAChR subunits combine togenerate unique NAChR subtypes, particularly in neuronal cells. Indeed,there is evidence that only certain NAChR subtypes may be involved indiseases such as Alzheimer's disease. Moreover, it is not clear whetherNAChRs from analogous tissues or cell types are similar across species.

Accordingly, there is a need for the isolation and characterization ofDNAs encoding each human neuronal NAChR subunit, recombinant cellscontaining such subunits and receptors prepared therefrom. In order tostudy the function of human neuronal AChRs and to obtaindisease-specific pharmacologically active agents, there is also a needto obtain isolated (preferably purified) human neuronal nicotinic AChRs,and isolated (preferably purified) human neuronal nicotinic AChRsubunits. In addition, there is also a need to develop assays toidentify such pharmacologically active agents.

The availability of such DNAs, cells, receptor subunits and receptorcompositions will eliminate the uncertainty of speculating as to humannNAChR structure and function based on predictions drawn from non-humannNAChR data, or human or non-human muscle or ganglia NAChR data.

Therefore, it is an object herein to isolate and characterize DNAencoding subunits of human neuronal nicotinic acetylcholine receptors.It is also an object herein to provide methods for recombinantproduction of human neuronal nicotinic acetylcholine receptor subunits.It is also an object herein to provide purified receptor subunits and toprovide methods for screening compounds to identify compounds thatmodulate the activity of human neuronal AChRs.

These and other objects will become apparent to those of skill in theart upon further study of the specification and claims.

BRIEF DESCRIPTION OF THE INVENTION

In accordance with the present invention, there are provided isolatedDNAs encoding novel human alpha and beta subunits of neuronal NAChRs. Inparticular, isolated DNA encoding human α₄, α₇, and β₄ subunits ofneuronal NAChRs are provided. Messenger RNA and polypeptides encoded bythe above-described DNA are also provided.

Further in accordance with the present invention, there are providedrecombinant human neuronal nicotinic AChR subunits, including α₄, α₇,and β₄ subunits, as well as methods for the production thereof. Inaddition, recombinant human neuronal nicotinic acetylcholine receptorscontaining at least one human neuronal nicotinic AChR subunit are alsoprovided, as well as methods for the production thereof. Furtherprovided are recombinant neuronal nicotinic AChRs that contain a mixtureof one or more NAChR subunits encoded by a host cell, and one or morenNAChR subunits encoded by heterologous DNA or RNA (i.e., DNA or RNA asdescribed herein that has been introduced into the host cell), as wellas methods for the production thereof.

Plasmids containing DNA encoding the above-described subunits are alsoprovided. Recombinant cells containing the above-described DNA, mRNA orplasmids are also provided herein. Such cells are useful, for example,for replicating DNA, for producing human NAChR subunits and recombinantreceptors, and for producing cells that express receptors containing oneor more human subunits.

Also provided in accordance with the present invention are methods foridentifying cells that express functional nicotinic acetylcholinereceptors. Methods for identifying compounds which modulate the activityof NAChRs are also provided.

The DNA, mRNA, vectors, receptor subunits, receptor subunit combinationsand cells provided herein permit production of selected neuronalnicotinic AChR receptor subtypes and specific combinations thereof, aswell as antibodies to said receptor subunits. This provides a means toprepare synthetic or recombinant receptors and receptor subunits thatare substantially free of contamination from many other receptorproteins whose presence can interfere with analysis of a single NAChRsubtype. The availability of desired receptor subtypes makes it possibleto observe the effect of a drug substance on a particular receptorsubtype and to thereby perform initial in vitro screening of the drugsubstance in a test system that is specific for humans and specific fora human neuronal nicotinic AChR subtype.

The availability of subunit-specific antibodies makes possible theapplication of the technique of immunohistochemistry to monitor thedistribution and expression density of various subunits (e.g., in normalvs diseased brain tissue). Such antibodies could also be employed fordiagnostic and therapeutic applications.

The ability to screen drug substances in vitro to determine the effectof the drug on specific receptor compositions should permit thedevelopment and screening of receptor subtype-specific ordisease-specific drugs. Also, testing of single receptor subunits orspecific receptor subtype combinations with a variety of potentialagonists or antagonists provides additional information with respect tothe function and activity of the individual subunits and should lead tothe identification and design of compounds that are capable of veryspecific interaction with one or more of the receptor subunits orreceptor subtypes. The resulting drugs should exhibit fewer unwantedside effects than drugs identified by screening with cells that expressa variety of subtypes.

Further in relation to drug development and therapeutic treatment ofvarious disease states, the availability of DNAs encoding human nNAChRsubunits enables identification of any alterations in such genes (e.g.,mutations) which may correlate with the occurrence of certain diseasestates. In addition, the creation of animal models of such diseasestates becomes possible, by specifically introducing such mutations intosynthetic DNA sequences which can then be introduced into laboratoryanimals or in vitro assay systems to determine the effects thereof.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 presents a restriction map of two pCMV promoter-based vectors,pCMV-T7-2 and pCMV-T7-3.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, we have isolated andcharacterized DNAs encoding novel human alpha and beta subunits ofneuronal NAChRs. Specifically, isolated DNAs encoding human α₄, α₇, andβ₄ subunits of neuronal NAChRs are described herein. Recombinantmessenger RNA (mRNA) and recombinant polypeptides encoded by theabove-described DNA are also provided.

As used herein, isolated (or substantially pure) as a modifier of DNA,RNA, polypeptides or proteins means that the DNA, RNA, polypeptides orproteins so designated have been separated from their in vivo cellularenvironments through the efforts of human beings. Thus as used herein,isolated (or substantially pure) DNA refers to DNAs purified accordingto standard techniques employed by those skilled in the art (see, e.g.,Maniatis et al.(1982) Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

Similarly, as used herein, "recombinant" as a modifier of DNA, RNA,polypeptides or proteins means that the DNA, RNA, polypeptides orproteins so designated have been prepared by the efforts of humanbeings, e.g., by cloning, recombinant expression, and the like. Thus asused herein, recombinant proteins, for example, refers to proteinsproduced by a recombinant host, expressing DNAs which have been added tothat host through the efforts of human beings.

As used herein, a human alpha subunit gene is a gene that encodes analpha subunit of a human neuronal nicotinic acetylcholine receptor. Thealpha subunit is a subunit of the NAChR to which ACh binds. Assignmentof the name "alpha" to a putative nNAChR subunit, according to Deneriset al. Tips (1991) 12:34-40! is based on the conservation of adjacentcysteine residues in the presumed extracellular domain of the subunitthat are the homologues of cysteines 192 and 193 of the Torpedo alphasubunit (see Noda et al. (1982) Nature 299:793-797). As used herein, analpha subunit subtype refers to a human nNAChR subunit that is encodedby DNA that hybridizes under high stringency conditions to at least oneof the nNAChR alpha subunit-encoding DNAs (or deposited clones)disclosed herein. An alpha subunit also binds to ACh under physiologicalconditions and at physiological concentrations and, in the optionalpresence of a beta subunit (i.e., some alpha subunits are functionalalone, while others require the presence of a beta subunit), generallyforms a functional AChR as assessed by methods described herein or knownto those of skill in this art.

Also contemplated are alpha subunits encoded by DNAs that encode alphasubunits as defined above, but that by virtue of degeneracy of thegenetic code do not necessarily hybridize to the disclosed DNA ordeposited clones under specified hybridization conditions. Such subunitsalso form a functional receptor, as assessed by the methods describedherein or known to those of skill in the art, generally with one or morebeta subunit subtypes. Typically, unless an alpha subunit is encoded byRNA that arises from alternative splicing (i.e., a splice variant),alpha-encoding DNA and the alpha subunit encoded thereby sharesubstantial sequence homology with at least one of the alpha subunitDNAs (and proteins encoded thereby) described or deposited herein. It isunderstood that DNA or RNA encoding a splice variant may overall shareless than 90% homology with the DNA or RNA provided herein, but includeregions of nearly 100% homology to a DNA fragment or deposited clonedescribed herein, and encode an open reading frame that includes startand stop codons and encodes a functional alpha subunit.

As used herein, a splice variant refers to variant NAChRsubunit-encoding nucleic acid(s) produced by differential processing ofprimary transcript(s) of genomic DNA, resulting in the production ofmore than one type of mRNA. cDNA derived from differentially processedgenomic DNA will encode NAChR subunits that have regions of completeamino acid identity and regions having different amino acid sequences.Thus, the same genomic sequence can lead to the production of multiple,related mRNAs and proteins. Both the resulting mRNAs and proteins arereferred to herein as "splice variants".

Stringency of hybridization is used herein to refer to conditions underwhich polynucleic acid hybrids are stable. As known to those of skill inthe art, the stability of hybrids is reflected in the meltingtemperature (T_(m)) of the hybrids. T_(m) can be approximated by theformula:

    81.5° C.-16.6(log.sub.10  Na.sup.+ !)+0.41(%G+C)-600/1,

where 1 is the length of the hybrids in nucleotides. T_(m) decreasesapproximately 1°-1.5° C. with every 1% decrease in sequence homology. Ingeneral, the stability of a hybrid is a function of sodium ionconcentration and temperature. Typically, the hybridization reaction isperformed under conditions of lower stringency, followed by washes ofvarying, but higher, stringency. Reference to hybridization stringencyrelates to such washing conditions. Thus, as used herein:

(1) HIGH STRINGENCY refers to conditions that permit hybridization ofonly those nucleic acid sequences that form stable hybrids in 0.018MNaCl at 65° C. (i.e., if a hybrid is not stable in 0.018M NaCl at 65°C., it will not be stable under high stringency conditions, ascontemplated herein). High stringency conditions can be provided, forexample, by hybridization in 50% formamide, 5× Denhardt's solution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.1× SSPE, and 0.1% SDSat 65° C.;

(2) MODERATE STRINGENCY refers to conditions equivalent to hybridizationin 50% formamide, 5× Denhardt's solution, 5× SSPE, 0.2% SDS at 42° C.,followed by washing in 0.2× SSPE, 0.2% SDS, at 65° C.; and

(3) LOW STRINGENCY refers to conditions equivalent to hybridization in10% formamide, 5× Denhardt's solution, 6× SSPE, 0.2% SDS, followed bywashing in 1× SSPE, 0.2% SDS, at 50° C.

It is understood that these conditions may be duplicated using a varietyof buffers and temperatures and that they are not necessarily precise.

Denhardt's solution and SSPE (see, e.g., Sambrook, Fritsch, andManiatis, in: Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratory Press, 1989) are well known to those of skill in the art asare other suitable hybridization buffers. For example, SSPE is pH 7.4phosphate-buffered 0.18M NaCl. SSPE can be prepared, for example, as a20× stock solution by dissolving 175.3 g of NaCl, 27.6 g of NaH₂ PO₄ and7.4 g EDTA in 800 ml of water, adjusting the pH to 7.4, and then addingwater to 1 liter. Denhardt's solution (see, Denhardt (1966) Biochem.Biophys. Res. Commun. 23:641) can be prepared, for example, as a 50×stock solution by mixing 5 g Ficoll (Type 400, Pharmacia LKBBiotechnology, INC., Piscataway N.J.), 5 g of polyvinylpyrrolidone, 5 gbovine serum albumin (Fraction V; Sigma, St. Louis Mo.) water to 500 mland filtering to remove particulate matter.

The phrase "substantial sequence homology" is used herein in referenceto the nucleotide sequence of DNA, the ribonucleotide sequence of RNA,or the amino acid sequence of protein, that have slight andnon-consequential sequence variations from the actual sequencesdisclosed herein. Species having substantial sequence homology areconsidered to be equivalent to the disclosed sequences and as such arewithin the scope of the appended claims. In this regard, "slight andnon-consequential sequence variations" mean that "homologous" sequences,i.e., sequences that have substantial homology with the DNA, RNA, orproteins disclosed and claimed herein, are functionally equivalent tothe sequences disclosed and claimed herein. Functionally equivalentsequences will function in substantially the same manner to producesubstantially the same compositions as the nucleic acid and amino acidcompositions disclosed and claimed herein. In particular, functionallyequivalent DNAs encode proteins that are the same as those disclosedherein or that have conservative amino acid variations, such assubstitution of a non-polar residue for another non-polar residue or acharged residue for a similarly charged residue. These changes includethose recognized by those of skill in the art as those that do notsubstantially alter the tertiary structure of the protein.

In practice, the term substantially the same sequence means that DNA orRNA encoding two proteins hybridize under conditions of high stringencyand encode proteins that have the same sequence of amino acids or havechanges in sequence that do not alter their structure or function. Asused herein, substantially identical sequences of nucleotides share atleast about 90% identity, and substantially identical amino acidsequences share more than 95% amino acid identity. It is recognized,however, that proteins (and DNA or mRNA encoding such proteins)containing less than the above-described level of homology arising assplice variants or that are modified by conservative amino acidsubstitutions (or substitution of degenerate codons) are contemplated tobe within the scope of the present invention.

As used herein, "α₄ subunit DNA" refers to DNA encoding a neuronalnicotinic acetylcholine receptor subunit of the same name. Such DNA canbe characterized in a number of ways, for example

said DNA may encode the amino acid sequence set forth in SEQ.ID.No:6, or

said DNA may encode the amino acid sequence encoded by clone HnAChRα4.2,deposited under ATCC Accession No. 69239, or

the 5' nucleotides of said DNA may encode the amino acid sequenceencoded by clone HnAChRα4.1, deposited under ATCC Accession No. 69152.

Presently preferred α₄ -encoding DNAs can be characterized as follows

said DNA may hybridize to the coding sequence set forth in SEQ.ID.No:5(preferably to substantially the entire coding sequence thereof, i.e.,nucleotides 184-2067) under high stringency conditions, or

said DNA may hybridize under high stringency conditions to the sequence(preferably to substantially the entire sequence) of the α₄ -encodinginsert of clone HnAChRα4.2, deposited under ATCC Accession No. 69239, or

the 5' nucleotides of said DNA may hybridize under high stringencyconditions to the sequence of the α₄ -encoding insert of cloneHnAChRα4.1, deposited under ATCC Accession No. 69152.

Especially preferred α₄ -encoding DNAs of the invention arecharacterized as follows

DNA having substantially the same nucleotide sequence as the codingregion set forth in SEQ.ID.No:5 (i.e., nucleotides 184-2067 thereof), or

DNA having substantially the same nucleotide sequence as the α₄-encoding insert of clone HnAChRα4.2, deposited under ATCC Accession No.69239, or

the 5' nucleotides of said DNA have substantially the same sequence asthe α₄ -encoding insert of clone HnAChRα4.1, deposited under ATCCAccession No. 69152.

Typically, unless an α₄ subunit arises as a splice variant, α₄ -encodingDNA will share substantial sequence homology (i.e., greater than about90%), with the α₄ DNAs described or deposited herein. DNA or RNAencoding a splice variant may share less than 90% overall sequencehomology with the DNA or RNA provided herein, but such a splice variantwould include regions of nearly 100% homology to the above-describedDNAs.

As used herein, "α₇ subunit DNA" refers to DNA encoding a neuronalnicotinic acetylcholine receptor subunit of the same name. Such DNA canbe characterized in a number of ways, for example, the nucleotides ofsaid DNA may encode the amino acid sequence set forth in SEQ.ID.No:8.Presently preferred α₇ -encoding DNAs can be characterized as DNA whichhybridizes under high stringency conditions to the coding sequence setforth in SEQ.ID.No:7 (preferably to substantially the entire codingsequence thereof, i.e., nucleotides 73-1581). Especially preferred α₇-encoding DNAs of the invention are characterized as havingsubstantially the same nucleotide sequence as the coding sequence setforth in SEQ.ID.No:7 (i.e., nucleotides 73-1581 thereof).

Typically, unless an α₇ subunit arises as a splice variant, α₇ -encodingDNA will share substantial sequence homology (greater than about 90%)with the α₇ DNAs described or deposited herein. DNA or RNA encoding asplice variant may share less than 90% overall sequence homology withthe DNA or RNA provided herein, but such DNA would include regions ofnearly 100% homology to the above-described DNA.

The α₇ subunits derived from the above-described DNA are expected tobind to the neurotoxin α-bungarotoxin (α-bgtx). The activity of AChRsthat contain α7 subunits should be inhibited upon interaction withα-bgtx. Amino acid residues 210 through 217, as set forth inSEQ.ID.No:8, are believed to be important elements in the binding ofα-bgtx (see, for example, Chargeux et al. Trends Pharmacol Sci. (1992)13:299-301).

As used herein, a human beta subunit gene is a gene that encodes a betasubunit of a human neuronal nicotinic acetylcholine receptor. Assignmentof the name "beta" to a putative nNAChR subunit, according to Deneris etal. supra, is based on the lack of adjacent cysteine residues (which arecharacteristic of alpha subunits). The beta subunit is frequentlyreferred to as the structural NAChR subunit (although it is possiblethat beta subunits also have ACh binding properties). Combination ofbeta subunit(s) with appropriate alpha subunit(s) leads to the formationof a functional receptor. As used herein, a beta subunit subtype refersto a nNAChR subunit that is encoded by DNA that hybridizes under highstringency conditions to at least one of the nNAChR-encoding DNAs (ordeposited clones) disclosed herein. A beta subunit forms a functionalNAChR, as assessed by methods described herein or known to those ofskill in this art, with appropriate alpha subunit subtype(s).

Also contemplated are beta subunits encoded by DNAs that encode betasubunits as defined above, but that by virtue of degeneracy of thegenetic code do not necessarily hybridize to the disclosed DNA ordeposited clones under the specified hybridization conditions. Suchsubunits also form functional receptors, as assessed by the methodsdescribed herein or known to those of skill in the art, in combinationwith appropriate alpha subunit subtype(s). Typically, unless a betasubunit is encoded by RNA that arises as a splice variant, beta-encodingDNA and the beta subunit encoded thereby share substantial sequencehomology with the beta-encoding DNA and beta subunit protein describedherein. It is understood that DNA or RNA encoding a splice variant mayshare less than 90% overall homology with the DNA or RNA providedherein, but such DNA will include regions of nearly 100% homology to theDNA described herein.

As used herein, "β₄ subunit DNA" refers to DNA encoding a neuronalnicotinic acetylcholine receptor subunit of the same name. Such DNA canbe characterized in a number of ways, for example, the nucleotides ofsaid DNA may encode the amino acid sequence set forth in SEQ.ID.No:12.Presently preferred β₄ -encoding DNAs can be characterized as DNA whichhybridizes under high stringency conditions to the coding sequence setforth in SEQ.ID.No:11 (preferably to substantially the entire codingsequence thereof, i.e., nucleotides 87-1583). Especially preferred β₄-encoding DNAs of the invention are characterized as havingsubstantially the same nucleotide sequence as set forth in SEQ.ID.No:11.

Typically, unless a β₄ subunit arises as a splice variant, β₄ -encodingDNA will share substantial sequence homology (greater than about 90%)with the β₄ DNAs described or deposited herein. DNA or RNA encoding asplice variant may share less than 90% overall sequence homology withthe DNA or RNA provided herein, but such DNA would include regions ofnearly 100% homology to the above-described DNA.

DNA encoding human neuronal nicotinic AChR alpha and beta subunits maybe isolated by screening suitable human cDNA or human genomic librariesunder suitable hybridization conditions with DNA disclosed herein(including nucleotides derived from any of SEQ.ID.Nos:5, 7, 9 or 11, orwith any of the deposited clones referred to herein (e.g., ATCCaccession no. 69239 or 69152). Suitable libraries can be prepared fromneuronal tissue samples, hippocampus tissue, or cell lines, such as thehuman neuroblastoma cell line IMR32 (ATCC Accession No. CCL127), and thelike. The library is preferably screened with a portion of DNA includingthe entire subunit-encoding sequence thereof, or the library may bescreened with a suitable probe.

As used herein, a probe is single-stranded DNA or RNA that has asequence of nucleotides that includes at least 14 contiguous bases thatare the same as (or the complement of) any 14 bases set forth in any ofSEQ.ID.Nos:1, 3, 5, 7, 9, or 11, or in the subunit encoding DNA in anyof the deposited clones described herein (e.g., ATCC accession no. 69239or 69152). Preferred regions from which to construct probes include 5'and/or 3' coding sequences, sequences predicted to encode transmembranedomains, sequences predicted to encode the cytoplasmic loop, signalsequences, acetylcholine (ACh) and α-bungarotoxin (α-bgtx) bindingsites, and the like. Amino acids 210-220 are typically involved in AChand α-bgtx binding. The approximate amino acid residues which comprisesuch regions for other preferred probes are set forth in the followingtable:

    __________________________________________________________________________    Subunit                                                                           Signal Sequence                                                                       TMD1*                                                                              TMD2 TMD3 TMD4 Cytoplasmic Loop                              __________________________________________________________________________    α.sub.2                                                                     1-55    264-289                                                                            297-320                                                                            326-350                                                                            444-515                                                                            351-443                                       α.sub.3                                                                     1-30    240-265                                                                            273-296                                                                            302-326                                                                            459-480                                                                            327-458                                       α.sub.4                                                                     1-33    241-269                                                                            275-289                                                                            303-330                                                                            593-618                                                                            594-617                                       α.sub.7                                                                     1-23    229-256                                                                            262-284                                                                            290-317                                                                            462-487                                                                            318-461                                       β.sub.2                                                                      1-25    234-259                                                                            267-288                                                                            295-320                                                                            453-477                                                                            321-452                                       β.sub.4                                                                      1-23    234-258                                                                            264-285                                                                            290-319                                                                            454-478                                                                            320-453                                       __________________________________________________________________________     *TMD = transmembrane domain                                              

Alternatively, portions of the DNA can be used as primers to amplifyselected fragments in a particular library.

After screening the library, positive clones are identified by detectinga hybridization signal; the identified clones are characterized byrestriction enzyme mapping and/or DNA sequence analysis, and thenexamined, by comparison with the sequences set forth herein or with thedeposited clones described herein, to ascertain whether they include DNAencoding a complete alpha or beta subunit. If the selected clones areincomplete, they may be used to rescreen the same or a different libraryto obtain overlapping clones. If desired, the library can be rescreenedwith positive clones until overlapping clones that encode an entirealpha or beta subunit are obtained. If the library is a cDNA library,then the overlapping clones will include an open reading frame. If thelibrary is genomic, then the overlapping clones may include exons andintrons. In both instances, complete clones may be identified bycomparison with the DNA and encoded proteins provided herein.

Complementary DNA clones encoding various subtypes of human nNAChR alphaand beta subunits have been isolated. Each subtype of the subunitappears to be encoded by a different gene. The DNA clones providedherein may be used to isolate genomic clones encoding each subtype andto isolate any splice variants by screening libraries prepared fromdifferent neural tissues. Nucleic acid amplification techniques, whichare well known in the art, can be used to locate splice variants ofhuman NAChR subunits. This is accomplished by employing oligonucleotidesbased on DNA sequences surrounding divergent sequence(s) as primers foramplifying human RNA or genomic DNA. Size and sequence determinations ofthe amplification products can reveal the existence of splice variants.Furthermore, isolation of human genomic DNA sequences by hybridizationcan yield DNA containing multiple exons, separated by introns thatcorrespond to different splice variants of transcripts encoding humanNAChR subunits.

It has been found that not all subunit subtypes are expressed in allneural tissues or in all portions of the brain. Thus, in order toisolate cDNA encoding particular subunit subtypes or splice variants ofsuch subtypes, it is preferable to screen libraries prepared fromdifferent neuronal or neural tissues. Preferred libraries for obtainingDNA encoding each subunit include: hippocampus to isolate human α₄ - andα₅ -encoding DNA; IMR32 to isolate human α₃ -, α₅ -, α₇ - and β₄-encoding DNA, thalamus to isolate α₂ and β₂ -encoding DNA; and thelike.

It appears that the distribution of expression of human neuronalnicotinic AChRs differs from the distribution of such receptors in rat.For example, RNA encoding the rat α₄ subunit is abundant in ratthalamus, but is not abundant in rat hippocampus (see, e.g., Wada et al.(1989) J. Comp. Neurol 284:314-335). No α₄ -encoding clones could beobtained, however, from a human thalamus library. Instead, human α₄clones were ultimately obtained from a human hippocampus library. Thus,the distribution of α₄ nNAChR subunit in humans and rats appears to bequite different.

Rat α₃ subunit appears to be a CNS-associated subunit that is abundantlyexpressed in the thalamus and weakly expressed in the brain stem (see,e.g., Boulter et al. (1986) Nature 319:368-374; Boulter et al. (1987)Proc. Natl. Acad. Sci. USA 84:7763-7767; and Wada et al. (1989) J. Comp.Neurol 284:314-335). In efforts to clone DNA encoding the humannicotinic AChR α₃ subunit, however, several human libraries, including athalamus library, were unsuccessfully screened. Surprisingly, clonesencoding human α₃ subunit were ultimately obtained from a brain stemlibrary and from IMR32 cells that reportedly express few, if any,functional nicotinic acetylcholine receptors (see, e.g., Gotti et al.((1986) Biochem. Biophys. Res. Commun. 137: 1141-1147, and Clementi etal. (1986) J. Neurochem. 47: 291-297).

Rat α₇ subunit transcript reportedly is abundantly expressed in thehippocampus (see Seguela et al. (1993) J. Neurosci. 13:596-604). Effortsto clone DNA encoding a human α₇ subunit from a human hippocampuslibrary (1×10⁶ recombinants) were unsuccessful. Surprisingly, clonesencoding a human NAChR α₇ subunit were ultimately obtained from an IMR32cell cDNA library.

The above-described nucleotide sequences can be incorporated intovectors for further manipulation. As used herein, vector (or plasmid)refers to discrete elements that are used to introduce heterologous DNAinto cells for either expression or replication thereof. Selection anduse of such vehicles are well within the level of skill of the art.

An expression vector includes vectors capable of expressing DNAs thatare operatively linked with regulatory sequences, such as promoterregions, that are capable of affecting expression of such DNA fragments.Thus, an expression vector refers to a recombinant DNA or RNA construct,such as a plasmid, a phage, recombinant virus or other vector that, uponintroduction into an appropriate host cell, results in expression of thecloned DNA. Appropriate expression vectors are well known to those ofskill in the art and include those that are replicable in eukaryoticcells and/or prokaryotic cells and those that remain episomal or thosewhich integrate into the host cell genome. Presently preferred plasmidsfor expression of invention AChR subunits in eukaryotic host cells,particularly mammalian cells, include cytomegalovirus (CMV)promoter-containing vectors such as pCMV, pcDNA1, and the like.

As used herein, a promoter region refers to a segment of DNA thatcontrols transcription of DNA to which it is operatively linked. Thepromoter region includes specific sequences that are sufficient for RNApolymerase recognition, binding and transcription initiation. Thisportion of the promoter region is referred to as the promoter. Inaddition, the promoter region includes sequences that modulate thisrecognition, binding and transcription initiation activity of RNApolymerase. These sequences may be cis acting or may be responsive totrans acting factors. Promoters, depending upon the nature of theregulation, may be constitutive or regulated. Exemplary promoterscontemplated for use in the practice of the present invention includethe SV40 early promoter, the cytomegalovirus (CMV) promoter, the mousemammary tumor virus (MMTV) steroid-inducible promoter, Moloney murineleukemia virus (MMLV) promoter, and the like.

As used herein, the term "operatively linked" refers to the functionalrelationship of DNA with regulatory and effector sequences ofnucleotides, such as promoters, enhancers, transcriptional andtranslational stop sites, and other signal sequences. For example,operative linkage of DNA to a promoter refers to the physical andfunctional relationship between the DNA and the promoter such that thetranscription of such DNA is initiated from the promoter by an RNApolymerase that specifically recognizes, binds to and transcribes theDNA. In order to optimize expression and/or in vitro transcription, itmay be necessary to remove or alter 5' untranslated portions of theclones to remove extra, potential alternative translation initiation(i.e., start) codons or other sequences that interfere with or reduceexpression, either at the level of transcription or translation.Alternatively, consensus ribosome binding sites (see, for example, Kozak(1991) J. Biol. Chem. 266:19867-19870) can be inserted immediately 5' ofthe start codon to enhance expression. The desirability of (or need for)such modification may be empirically determined.

As used herein, expression refers to the process by which polynucleicacids are transcribed into mRNA and translated into peptides,polypeptides, or proteins. If the polynucleic acid is derived fromgenomic DNA, expression may, if an appropriate eukaryotic host cell ororganism is selected, include splicing of the mRNA.

Particularly preferred vectors for transfection of mammalian cells arethe pSV2dhfr expression vectors, which contain the SV40 early promoter,mouse dhfr gene, SV40 polyadenylation and splice sites and sequencesnecessary for maintaining the vector in bacteria, cytomegalovirus (CMV)promoter-based vectors such as pCDNA1 (Invitrogen, San Diego, Calif.),and MMTV promoter-based vectors such as pMSG (Catalog No. 27-4506-01from Pharmacia, Piscataway, N.J.).

Full-length DNAs encoding human neuronal NAChR subunits have beeninserted into vector pCMV-T7, a pUC19-based mammalian cell expressionvector containing the CMV promoter/enhancer, SV40 splice/donor siteslocated immediately downstream of the promoter, a polylinker downstreamof the splice/donor sites, followed by an SV40 polyadenylation signal.Placement of NAChR subunit DNA between the CMV promoter and SV40polyadenylation signal provides for constitutive expression of theforeign DNA in a mammalian host cell transfected with the construct. Forinducible expression of human NAChR subunit-encoding DNA in a mammaliancell, the DNA can be inserted into a plasmid such as PMSG. This plasmidcontains the mouse mammary tumor virus (MMTV) promoter forsteroid-inducible expression of operatively associated foreign DNA. Ifthe host cell does not express endogenous glucocorticoid receptorsrequired for uptake of glucocorticoids (i.e., inducers of the MMTVpromoter) into the cell, it is necessary to additionally transfect thecell with DNA encoding the glucocorticoid receptor (ATCC accession no.67200). Full-length human DNA clones encoding human α₃, α₄, α₇, β₂ andβ₄ have also been subcloned into pIBI24 (International Biotechnologies,Inc., New Haven, Conn.) or pCMV-T7-2 for synthesis of in vitrotranscripts.

In accordance with another embodiment of the present invention, thereare provided cells containing the above-described polynucleic acids(i.e., DNA or mRNA). Such host cells as bacterial, yeast and mammaliancells can be used for replicating DNA and producing nAChR subunit(s).Methods for constructing expression vectors, preparing in vitrotranscripts, transfecting DNA into mammalian cells, injecting oocytes,and performing electrophysiological and other analyses for assessingreceptor expression and function as described herein are also describedin PCT Application Nos. PCT/US91/02311, PCT/US91/05625 andPCT/US92/11090, and in co-pending U.S. application Ser. Nos. 07/504,455,07/563,751 and 07/812,254. The subject matter of these applications arehereby incorporated by reference herein in their entirety.

Incorporation of cloned DNA into a suitable expression vector,transfection of eukaryotic cells with a plasmid vector or a combinationof plasmid vectors, each encoding one or more distinct genes or withlinear DNA, and selection of transfected cells are well known in the art(see, e.g., Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press).Heterologous DNA may be introduced into host cells by any method knownto those of skill in the art, such as transfection with a vectorencoding the heterologous DNA by CaPO₄ precipitation (see, e.g., Wigleret al. (1979) Proc. Natl. Acad. Sci. 76:1373-1376). Recombinant cellscan then be cultured under conditions whereby the subunit(s) encoded bythe DNA is (are) expressed. Preferred cells include mammalian cells(e.g., HEK 293, CHO and Ltk⁻ cells), yeast cells (e.g., methylotrophicyeast cells, such as Pichia pastoris), bacterial cells (e.g.,Escherichia coli), and the like.

While the DNA provided herein may be expressed in any eukaryotic cell,including yeast cells (such as, for example, P. pastoris (see U.S. Pat.Nos. 4,882,279, 4,837,148, 4,929,555 and 4,855,231), Saccharomycescerevisiae, Candida tropicalis, Hansenula polymorpha, and the like),mammalian expression systems, including commercially available systemsand other such systems known to those of skill in the art, forexpression of DNA encoding the human neuronal nicotinic AChR subunitsprovided herein are presently preferred. Xenopus oocytes are preferredfor expression of RNA transcripts of the DNA.

In preferred embodiments, DNA is ligated into a vector, and introducedinto suitable host cells to produce transformed cell lines that expressa specific human nNAChR receptor subtype, or specific combinations ofsubtypes. The resulting cell lines can then be produced in quantity forreproducible quantitative analysis of the effects of drugs on receptorfunction. In other embodiments, mRNA may be produced by in vitrotranscription of DNA encoding each subunit. This mRNA, either from asingle subunit clone or from a combination of clones, can then beinjected into Xenopus oocytes where the RNA directs the synthesis of thehuman receptor subunits, which then form functional receptors.Alternatively, the subunit-encoding DNA can be directly injected intooocytes for expression of functional receptors. The transfectedmammalian cells or injected oocytes may then be used in the methods ofdrug screening provided herein.

Cloned full-length DNA encoding any of the subunits of human neuronalnicotinic AChR may be introduced into a plasmid vector for expression ina eukaryotic cell. Such DNA may be genomic DNA or cDNA. Host cells maybe transfected with one or a combination of plasmids, each of whichencodes at least one human neuronal nicotinic AChR subunit.

Eukaryotic cells in which DNA or RNA may be introduced include any cellsthat are transfectable by such DNA or RNA or into which such DNA or RNAmay be injected. Preferred cells are those that can be transiently orstably transfected and also express the DNA and RNA. Presently mostpreferred cells are those that can form recombinant or heterologoushuman neuronal nicotinic AChRs comprising one or more subunits encodedby the heterologous DNA. Such cells may be identified empirically orselected from among those known to be readily transfected or injected.

Exemplary cells for introducing DNA include cells of mammalian origin(e.g., COS cells, mouse L cells, Chinese hamster ovary (CHO) cells,human embryonic kidney cells, African green monkey cells and other suchcells known to those of skill in the art), amphibian cells (e.g.,Xenopus laevis oocytes), yeast cells (e.g., Saccharomyces cerevisiae,Pichia pastoris), and the like. Exemplary cells for expressing injectedRNA transcripts include Xenopus laevis oocytes. Cells that are preferredfor transfection of DNA are known to those of skill in the art or may beempirically identified, and include HEK 293 (which are available fromATCC under accession #CRL 1573); Ltk⁻ cells (which are available fromATCC under accession #CCL1.3); COS-7 cells (which are available fromATCC under accession #CRL 1651); and DG44 cells (dhrf⁻ CHO cells; see,e.g., Urlaub et al. (1986) Cell. Molec. Genet. 12: 555). Presentlypreferred cells include DG44 cells and HEK 293 cells, particularly HEK293 cells that have been adapted for growth in suspension and that canbe frozen in liquid nitrogen and then thawed and regrown. HEK 293 cellsare described, for example, in U.S. Pat. No. 5,024,939 to Gorman (see,also, Stillman et al. (1985) Mol. Cell. Biol. 5:2051-2060).

DNA may be stably incorporated into cells or may be transientlyintroduced using methods known in the art. Stably transfected mammaliancells may be prepared by transfecting cells with an expression vectorhaving a selectable marker gene (such as, for example, the gene forthymidine kinase, dihydrofolate reductase, neomycin resistance, and thelike), and growing the transfected cells under conditions selective forcells expressing the marker gene. To produce such cells, the cellsshould be transfected with a sufficient concentration ofsubunit-encoding nucleic acids to form human neuronal nicotinic AChRsthat contain the human subunits encoded by heterologous DNA. The preciseamounts and ratios of DNA encoding the subunits may be empiricallydetermined and optimized for a particular combination of subunits, cellsand assay conditions. Recombinant cells that express neuronal nicotinicAChR containing subunits encoded only by the heterologous DNA or RNA areespecially preferred.

Heterologous DNA may be maintained in the cell as an episomal element ormay be integrated into chromosomal DNA of the cell. The resultingrecombinant cells may then be cultured or subcultured (or passaged, inthe case of mammalian cells) from such a culture or a subculturethereof. Methods for transfection, injection and culturing recombinantcells are known to the skilled artisan. Similarly, the human neuronalnicotinic AChR subunits may be purified using protein purificationmethods known to those of skill in the art. For example, antibodies orother ligands that specifically bind to one or more of the subunits maybe used for affinity purification of the subunit or human neuronalnicotinic AChRs containing the subunits.

In accordance with one embodiment of the present invention, methods forproducing cells that express human neuronal nicotinic AChR subunits andfunctional receptors are also provided. In one such method, host cellsare transfected with DNA encoding at least one alpha subunit of aneuronal nicotinic acetylcholine receptor and at least one beta subunitof a neuronal nicotinic acetylcholine receptor. Using methods such asnorthern blot or slot blot analysis, transfected cells that containalpha and/or beta subunit encoding DNA or RNA can be selected.Transfected cells are also analyzed to identify those that express NAChRprotein. Analysis can be carried out, for example, by measuring theability of cells to bind acetylcholine, nicotine, or a nicotine agonist,compared to the nicotine binding ability of untransfected host cells orother suitable control cells, by electrophysiologically monitoring thecurrents through the cell membrane in response to a nicotine agonist,and the like.

In particularly preferred aspects, eukaryotic cells which containheterologous DNAs express such DNA and form recombinant functionalneuronal nicotinic AChR(s). In more preferred aspects, recombinantneuronal nicotinic AChR activity is readily detectable because it is atype that is absent from the untransfected host cell or is of amagnitude not exhibited in the untransfected cell. Such cells thatcontain recombinant receptors could be prepared, for example, by causingcells transformed with DNA encoding the human neuronal nicotinic AChR α₃and β₄ subunits to express the corresponding proteins. The resultingsynthetic or recombinant receptor would contain only the α₃ and β₄nNAChR subunits. Such a receptor would be useful for a variety ofapplications, e.g., as part of an assay system free of the interferencesfrequently present in prior art assay systems employing non-humanreceptors or human tissue preparations. Furthermore, testing of singlereceptor subunits with a variety of potential agonists or antagonistswould provide additional information with respect to the function andactivity of the individual subunits. Such information may lead to theidentification of compounds which are capable of very specificinteraction with one or more of the receptor subunits. Such specificitymay prove of great value in medical application.

Thus, DNA encoding one or more human neuronal nicotinic AChR subunitsmay be introduced into suitable host cells (e.g., eukaryotic orprokaryotic cells) for expression of individual subunits and functionalNAChRs. Preferably combinations of alpha and beta subunits may beintroduced into cells: such combinations include combinations of any oneor more of al, α₁, α₂, α₃, α₄, α₅ and α₇ with β₂ or β₄. Sequenceinformation for α₁ is presented in Biochem. Soc. Trans. (1989)17:219-220; sequence information for α₅ is presented in Proc. Natl.Acad. Sci. USA (1992) 89:1572-1576; and sequence information for α₂, α₃,α₄, α₇, β₂ and β₄ is presented in the Sequence Listing providedherewith. Presently preferred combinations of subunits include any oneor more of α₁, α₂, α₃ or α₅ with β₄ ; or α₄ or α₇ in combination witheither β₂ or β₄. It is recognized that some of the subunits may have iontransport function in the absence of additional subunits. For example,the α₇ subunit is functional in the absence of any added beta subunit.

As used herein, "α₂ subunit DNA" refers to DNA that encodes a humanneuronal nicotinic acetylcholine receptor subunit of the same name, andto DNA that hybridizes under conditions of high stringency to the DNA ofSEQ.ID.No:1, or to the DNA of deposited clone having ATCC Accession No.68277, or to DNA that encodes the amino acid sequence set forth inSEQ.ID.No:2. Typically, unless an α₂ subunit arises as a splice variant,an α₂ DNA shares substantial sequence homology (greater than about 90%)with the α₂ DNA described herein. DNA or RNA encoding a splice variantmay share less than 90% overall sequence homology with the DNA or RNAdescribed herein, but such a splice variant would include regions ofnearly 100% homology to the above-described DNA.

As used herein, "α₃ subunit DNA" refers to DNA that encodes a neuronalsubunit of the same name, and to DNA that hybridizes under conditions ofhigh stringency to the DNA of SEQ.ID.No:3, or to the DNA of depositedclone having ATCC Accession No. 68278, or to DNA that encodes the aminoacid sequence set forth in SEQ.ID.No:4 Typically, unless an α₃ arises asa splice variant, an α₃ DNA shares substantial sequence homology(greater than about 90%) with the α₃ DNA described herein. DNA or RNAencoding a splice variant may share less than 90% overall sequencehomology with the DNA or RNA provided herein, but such a splice variantwould include regions of nearly 100% homology to the above describedDNA.

As used herein, "α₅ subunit DNA" refers to DNA that encodes a humanneuronal nicotinic acetylcholine receptor subunit of the same name, asdescribed, for example, by Chini et al. (1992) Proc. Natl. Acad. Sci.U.S.A. 89:1572-1576.

As used herein, "β₂ subunit DNA" refers to DNA that encodes a neuronalsubunit of the same name and, to DNA that hybridizes under conditions ofhigh stringency to the DNA of SEQ.ID.No:9, or to the DNA of depositedclone HnAChβ62, having ATCC Accession No. 68279, or to DNA encoding theamino acid sequence set forth in SEQ.ID.No:10. Typically, unless a β₂subunit arises as a splice variant, a β₂ DNA shares substantial sequencehomology (greater than about 90%) with the β₂ DNA described herein. DNAor RNA encoding a splice variant may share overall less than 90%homology with the DNA or RNA provided herein, but such a splice variantwould include regions of nearly 100% homology to the above-describedDNA.

In certain embodiments, eukaryotic cells with heterologous humanneuronal nicotinic AChRs are produced by introducing into the cell afirst composition, which contains at least one RNA transcript that istranslated in the cell into a subunit of a human neuronal nicotinicAChR. In preferred embodiments, the subunits that are translated includean alpha subunit of a human neuronal nicotinic AChR. More preferably,the composition that is introduced contains an RNA transcript whichencodes an alpha subunit and also contains an RNA transcript whichencodes a beta subunit of a human neuronal nicotinic AChR. RNAtranscripts can be obtained from cells transfected with DNAs encodinghuman neuronal nicotinic acetylcholine receptor subunits or by in vitrotranscription of subunit-encoding DNAs. Methods for in vitrotranscription of cloned DNA and injection of the resulting mRNA intoeukaryotic cells are well known in the art. Amphibian oocytes areparticularly preferred for expression of in vitro transcripts of thehuman nNAChR DNA clones provided herein. See, for example, Dascal (1989)CRC Crit. Rev. Biochem. 22:317-387, for a review of the use of Xenopusoocytes to study ion channels.

Thus, pairwise (or stepwise) introduction of DNA or RNA encoding alphaand beta subtypes into cells is possible. The resulting cells may betested by the methods provided herein or known to those of skill in theart to detect functional AChR activity. Such testing will allow theidentification of pairs of alpha and beta subunit subtypes that producefunctional AChRs, as well as individual subunits that produce functionalAChRs.

As used herein, activity of a human neuronal nicotinic AChR refers toany activity characteristic of an NAChR. Such activity can typically bemeasured by one or more in vitro methods, and frequently corresponds toan in vivo activity of a human neuronal nicotinic AChR. Such activitymay be measured by any method known to those of skill in the art, suchas, for example, measuring the amount of current which flows through therecombinant channel in response to a stimulus.

Methods to determine the presence and/or activity of human neuronalnicotinic AChRs include assays that measure nicotine binding, ⁸⁶ Rbion-flux, Ca²⁺ influx, the electrophysiological response of cells, theelectrophysiological response of oocytes transfected with RNA from thecells, and the like. In particular, methods are provided herein for themeasurement or detection of an AChR-mediated response upon contact ofcells containing the DNA or mRNA with a test compound.

As used herein, a recombinant or heterologous human neuronal nicotinicAChR refers to a receptor that contains one or more subunits encoded byheterologous DNA that has been introduced into and expressed in cellscapable of expressing receptor protein. A recombinant human neuronalnicotinic AChR may also include subunits that are produced by DNAendogenous to the host cell. In certain embodiments, recombinant orheterologous human neuronal nicotinic AChR may contain only subunitsthat are encoded by heterologous DNA.

As used herein, heterologous or foreign DNA and RNA are usedinterchangeably and refer to DNA or RNA that does not occur naturally aspart of the genome of the cell in which it is present or to DNA or RNAwhich is found in a location or locations in the genome that differ fromthat in which it occurs in nature. Typically, heterologous or foreignDNA and RNA refers to DNA or RNA that is not endogenous to the host celland has been artificially introduced into the cell. Examples ofheterologous DNA include DNA that encodes a human neuronal nicotinicAChR subunit, DNA that encodes RNA or proteins that mediate or alterexpression of endogenous DNA by affecting transcription, translation, orother regulatable biochemical processes, and the like. The cell thatexpresses heterologous DNA may contain DNA encoding the same ordifferent expression products. Heterologous DNA need not be expressedand may be integrated into the host cell genome or maintainedepisomally.

Recombinant receptors on recombinant eukaryotic cell surfaces maycontain one or more subunits encoded by the DNA or mRNA encoding humanneuronal nicotinic AChR subunits, or may contain a mixture of subunitsencoded by the host cell and subunits encoded by heterologous DNA ormRNA. Recombinant receptors may be homogeneous or may be a mixture ofsubtypes. Mixtures of DNA or mRNA encoding receptors from variousspecies, such as rats and humans, may also be introduced into the cells.Thus, a cell can be prepared that expresses recombinant receptorscontaining only α₃ and β₄ subunits, or any other combination of alphaand beta subunits provided herein. For example, α₄ and/or α₇ subunits ofthe present invention can be co-expressed with β₂ and/or β₄ receptorsubunits; similarly, β₄ subunits according to the present invention canbe co-expressed with α₂, α₃, α₄, α₅ and/or α₇ receptor subunits. Asnoted previously, some of the nNAChR subunits may be capable of formingfunctional receptors in the absence of other subunits, thusco-expression is not always required to produce functional receptors.

As used herein, a functional neuronal nicotinic AChR is a receptor thatexhibits an activity of neuronal nicotinic AChRs as assessed by any invitro or in vivo assay disclosed herein or known to those of skill inthe art. Possession of any such activity that may be assessed by anymethod known to those of skill in the art and provided herein issufficient to designate a receptor as functional. Methods for detectingNAChR protein and/or activity include, for example, assays that measurenicotine binding, ⁸⁶ Rb ion-flux, Ca²⁺ influx, the electrophysiologicalresponse of cells containing heterologous DNA or mRNA encoding one ormore receptor subunit subtypes, and the like. Since all combinations ofalpha and beta subunits may not form functional receptors, numerouscombinations of alpha and beta subunits should be tested in order tofully characterize a particular subunit and cells which produce same.Thus, as used herein, "functional" with respect to a recombinant orheterologous human neuronal nicotinic AChR means that the receptorchannel is able to provide for and regulate entry of human neuronalnicotinic AChR-permeable ions, such as, for example, Na⁺, K⁺, Ca²⁺ orBa²⁺, in response to a stimulus and/or bind ligands with affinity forthe receptor. Preferably such human neuronal nicotinic AChR activity isdistinguishable, such as by electrophysiological, pharmacological andother means known to those of skill in the art, from any endogenousnicotinic AChR activity that may be produced by the host cell.

In accordance with a particular embodiment of the present invention,recombinant human neuronal nicotinic AChR-expressing mammalian cells oroocytes can be contacted with a test compound, and the modulatingeffect(s) thereof can then be evaluated by comparing the AChR-mediatedresponse in the presence and absence of test compound, or by comparingthe AChR-mediated response of test cells, or control cells (i.e., cellsthat do not express nNAChRs), to the presence of the compound.

As used herein, a compound or signal that "modulates the activity of aneuronal nicotinic AChR" refers to a compound or signal that alters theactivity of NAChR so that activity of the NAChR is different in thepresence of the compound or signal than in the absence of the compoundor signal. In particular, such compounds or signals include agonists andantagonists. The term agonist refers to a substance or signal, such asACh, that activates receptor function; and the term antagonist refers toa substance that interferes with receptor function. Typically, theeffect of an antagonist is observed as a blocking of activation by anagonist. Antagonists include competitive and non-competitiveantagonists. A competitive antagonist (or competitive blocker) interactswith or near the site specific for the agonist (e.g., ligand orneurotransmitter) for the same or closely situated site. Anon-competitive antagonist or blocker inactivates the functioning of thereceptor by interacting with a site other than the site that interactswith the agonist.

As understood by those of skill in the art, assay methods foridentifying compounds that modulate human neuronal nicotinic AChRactivity (e.g., agonists and antagonists) generally require comparisonto a control. One type of a "control" cell or "control" culture is acell or culture that is treated substantially the same as the cell orculture exposed to the test compound, except the control culture is notexposed to test compound. For example, in methods that use voltage clampelectrophysiological procedures, the same cell can be tested in thepresence and absence of test compound, by merely changing the externalsolution bathing the cell. Another type of "control" cell or "control"culture may be a cell or a culture of cells which are identical to thetransfected cells, except the cells employed for the control culture donot express functional human neuronal nicotinic AChRs. In thissituation, the response of test cell to test compound is compared to theresponse (or lack of response) of receptor-negative (control) cell totest compound, when cells or cultures of each type of cell are exposedto substantially the same reaction conditions in the presence ofcompound being assayed.

The functional recombinant human neuronal nicotinic AChR includes atleast an alpha subunit, or an alpha subunit and a beta subunit of ahuman neuronal nicotinic AChR. Eukaryotic cells expressing thesesubunits have been prepared by injection of RNA transcripts and bytransfection of DNA. Such cells have exhibited nicotinic AChR activityattributable to human neuronal nicotinic AChRs that contain one or moreof the heterologous human neuronal nicotinic AChR subunits. For example,Xenopus laevis oocytes that had been injected with in vitro transcriptsof the DNA encoding human neuronal nicotinic AChR α₃ and β₄ subunitsexhibited AChR agonist induced currents; whereas cells that had beeninjected with transcripts of either the α₃ or β₄ subunit alone did not.In addition, HEK 293 cells that had been co-transfected with DNAencoding human neuronal NAChR α₃ and β₄ subunits exhibited AChRagonist-induced increases in intracellular calcium concentration,whereas control HEK 293 cells (i.e., cells that had not been transfectedwith α₃ - and β₄ -encoding DNA) did not exhibit any AChR agonist-inducedincreases in intracellular calcium concentration.

With respect to measurement of the activity of functional heterologoushuman neuronal nicotinic AChRs, endogenous AChR activity and, ifdesired, activity of AChRs that contain a mixture of endogenous hostcell subunits and heterologous subunits, should, if possible, beinhibited to a significant extent by chemical, pharmacological andelectrophysiological means.

Deposits

The deposited clones have been deposited at the American Type CultureCollection (ATCC), 12301 Parklawn Drive, Rockville, Md., U.S.A. 20852,under the terms of the Budapest Treaty on the International Recognitionof Deposits of Microorganisms for Purposes of Patent Procedure and theRegulations promulgated under this Treaty. Samples of the depositedmaterial are and will be available to industrial property offices andother persons legally entitled to receive them under the terms of theTreaty and Regulations and otherwise in compliance with the patent lawsand regulations of the United States of America and all other nations orinternational organizations in which this application, or an applicationclaiming priority of this application, is filed or in which any patentgranted on any such application is granted. In particular, upon issuanceof a U.S. Patent based on this or any application claiming priority toor incorporating this application by reference thereto, all restrictionsupon availability of the deposited material will be irrevocably removed.

The invention will now be described in greater detail with reference tothe following non-limiting examples.

EXAMPLE 1 Isolation of DNA Encoding Human nNAChR Subunits

A. DNA Encoding a Human nNAChR β₄ Subunit

Random primers were used in synthesizing cDNA from RNA isolated from theIMR32 human neuroblastoma cell line (the cells had been treated withdibutyryl cAMP and bromodeoxyuridine prior to constructing the library).The library constructed from the cDNAs was screened with a fragment of arat nicotinic AChR β₄ subunit cDNA. Hybridization was performed at 42°C. in 5× SSPE, 5× Denhardt's solution, 50% formamide, 200 μg/ml herringsperm DNA and 0.2% SDS. Washes were performed in 0.1× SSPE, 0.2% SDS at65° C. Five clones were identified that hybridized to the probe.

The five clones were plaque-purified and characterized by restrictionenzyme mapping and DNA sequence analysis. The insert DNA of one of thefive clones contained the complete coding sequence of a β₄ subunit of ahuman nicotinic AChR (see nucleotides 87-1583 of SEQ.ID.No:11). Theamino acid sequence deduced from the nucleotide sequence of thefull-length clone has ˜81% identity with the amino acid sequence deducedfrom the rat nicotinic AChR β₄ subunit DNA. Several regions of thededuced rat and human β₄ amino acid sequences are notably dissimilar:amino acids 1-23 (the human sequence has only ˜36% identity with respectto the rat sequence), 352-416 (the human sequence has only ˜48% identitywith respect to the rat sequence), and 417-492 (the human sequence hasonly ˜78% identity with respect to the rat sequence). Furthermore, aminoacids 376-379 in the rat β₄ subunit are not contained in the human β₄subunit.

B. DNA Encoding a Human nNAChR α₇ Subunit

An amplified IMR32 cell cDNA library (1×10⁶ recombinants; cells treatedwith dibutyryl cAMP and bromodeoxyuridine) was screened with a fragmentof a rat nicotinic AChR α₇ subunit cDNA. The hybridization conditionswere identical to those described above for screening an IMR32 cell cDNAlibrary with the rat β₄ subunit DNA. Washes were performed in 0.2× SSPE,0.2% SDS at 65° C. Seven positive clones were identified byhybridization to the labeled rat DNA probe. Six of the clones wereplaque-purified and characterized by restriction enzyme mapping and DNAsequence analysis. One of the clones contains the complete codingsequence of a human AChR receptor α₇ subunit gene (see nucleotides73-1581 of SEQ.ID.No:7).

C. DNA Encoding a Human nNAChR α₄ Subunit

Random primers were used in synthesizing cDNA from RNA isolated fromhuman hippocampus tissue. cDNAs larger than 2.0 kb were inserted intothe λgt10 phage vector to create a cDNA library. Approximately 1×10⁶recombinants were screened with a fragment of a DNA encoding a ratnicotinic AChR α₄ subunit using the same hybridization and washingconditions as described above for screening an IMR32 cell cDNA libraryfor α₇ subunit cDNAs. Three clones hybridized strongly to the probe. Twoof these three clones, designated KEα4.1 and KEα4.2, have been depositedwith the American Type Culture Collection (ATCC, Rockville, Md.) andassigned accession nos. 69152 and 69239, respectively.

Characterization of the plaque-purified clones revealed that one of theclones, KEα4.2, contains the complete coding sequence of a humannicotinic AChR α4 subunit gene (coding sequence of this human α₄ subunitcDNA is provided as nucleotides 184-2067 in SEQ.ID.No:5). Comparison ofthe 5' ends of the coding sequences of the human and rat α4 subunitcDNAs reveals that the rat sequence contains an 18-nucleotide segmentthat is not present in the human sequence.

D. DNA Encoding Human nNAChR α₂, α₃, & β₂ Subunits

Plasmids containing DNA that encodes and/or that can be used to isolateDNA that encodes human neuronal nicotinic acetylcholine receptor α₂, α₃and β₂ subunits have been deposited with the American Type CultureCollection (ATCC). The clone names and deposit accession numbers are:

    ______________________________________                                        Subunit    Clone Name                                                                              ATCC Accession No.                                       ______________________________________                                        α.sub.2                                                                            HnAChRα2                                                                          68277                                                    α.sub.3                                                                            HnACHRα3                                                                          68278                                                    β.sub.2                                                                             HnAChRβ2                                                                           68279                                                    ______________________________________                                    

In addition, DNA sequences that encode full-length α₂, α₃ and β₂subunits are set forth in SEQ.ID.Nos:1, 3 and 9, respectively.

EXAMPLE 2 Preparation of Constructs for the Expression of RecombinantHuman Neuronal Nicotinic AChR Subunits

Isolated cDNAs encoding human neuronal nicotinic AChR subunits wereincorporated into vectors for use in expressing the subunits inmammalian host cells and for use in generating in vitro transcripts ofthe DNAs to be expressed in Xenopus oocytes. Several different vectorswere utilized in preparing the constructs as follows.

A. Construct for Expression of a Human nNAChR α₃ Subunit

DNA encoding a human neuronal nicotinic AChR α₃ subunit was subclonedinto the pCMV-T7-2 general expression vector to create pCMV-KEα3.Plasmid pCMV-T7-2 (see FIG. 1) is a pUC19-based vector that contains aCMV promoter/enhancer, SV40 splice donor/splice acceptor sites locatedimmediately downstream of the promoter, a T7 bacteriophage RNApolymerase promoter positioned downstream of the SV40 splice sites, anSV40 polyadenylation signal downstream of the T7 promoter, and apolylinker between the T7 promoter and the polyadenylation signal. Thisvector thus contains all the regulatory elements required for expressionof heterologous DNA in a mammalian host cell, wherein the heterologousDNA has been incorporated into the vector at the polylinker. Inaddition, because the T7 promoter is located just upstream of thepolylinker, this plasmid can be used for synthesis of in vitrotranscripts of heterologous DNA that has been subcloned into the vectorat the polylinker. FIG. 1 also shows a restriction map of pCMV-T7-3.This plasmid is identical to pCMV-T7-2 except that the restriction sitesin the polylinker are in the opposite order as compared to the order inwhich they occur in pCMV-T7-2.

A 1.7 kb SfiI (blunt-ended)/EcoRI DNA fragment containing nucleotides27-1759 of SEQ.ID.No:3 (i.e., the entire α₃ subunit coding sequence plus12 nucleotides of 5' untranslated sequence and 206 nucleotides of 3'untranslated sequence) was ligated to EcoRV/EcoRI-digested pCMV-T7-2 togenerate pCMV-KEα3. Plasmid pCMV-KEα3 was used for expression of the α₃subunit in mammalian cells and for generating in vitro transcripts ofthe α₃ subunit DNA.

B. Constructs for Expression of a Human nNAChR β₄ Subunit

A 1.9 kb EcoRI DNA fragment containing nucleotides 1-1915 ofSEQ.ID.No:11 (i.e., the entire β₄ subunit coding sequence plus 86nucleotides of 5' untranslated sequence and 332 nucleotides of 3'untranslated sequence) was ligated to EcoRI-digested pGEM7Zf(+) (PromegaCatalog #P2251; Madison, Wis.). The resulting construct, KEβ4.6/pGEM,contains the T7 bacteriophage RNA polymerase promoter in operativeassociation with two tandem β₄ subunit DNA inserts (in the sameorientation) and was used in generating in vitro transcripts of the DNA.

The same 1.9 kb EcoRI DNA fragment containing nucleotides 1-1915 ofSEQ.ID.No:11 was ligated as a single insert to EcoRI-digested pCMV-T7-3to generate pCMV-KEβ4. Plasmid pCMV-KEβ4 was used for expression of theβ₄ subunit in mammalian cells and for generating in vitro transcripts ofthe β₄ subunit DNA.

C. Constructs for Expression of a Human nNAChR α₇ Subunit

Two pCMV-T7-2-based constructs were prepared for use in recombinantexpression of a human neuronal nicotinic AChR α₇ subunit. The firstconstruct, pCMV-KEα7.3, was prepared by ligating a 1.9 kb XhoI DNAfragment containing nucleotides 1-1876 of SEQ.ID.No:7 (i.e., the entireα₇ subunit coding sequence plus 72 nucleotides of 5' untranslatedsequence and 295 nucleotides of 3' untranslated sequence) toSalI-digested pCMV-T7-3. The second construct, pCMV-KEα7, was preparedby replacing the 5' untranslated sequence of the 1.9 kb XhoI α₇ subunitDNA fragment described above with a consensus ribosome binding site(5'-GCCACC-3'; see Kozak (1987) Nucl. Acids Res. 15:8125-8148). Theresulting modified fragment was ligated as a 1.8 kb BglII/XhoI fragmentwith BglII/SalI-digested pCMV-T7-2 to generate pCMV-KEα7. Thus, inpCMV-KEα7, the translation initiation codon of the coding sequence ofthe α₇ subunit cDNA is preceded immediately by a consensus ribosomebinding site.

D. Constructs for Expression of a Human nNAChR β₂ Subunit

DNA fragments encoding portions of a human neuronal nicotinic AChR β₂subunit were ligated together to generate a full-length β₂ subunitcoding sequence contained in plasmid pIBI24 (InternationalBiotechnologies, Inc. (IBI), New Haven, Conn.). The resulting construct,Hβ2.1F, contains nucleotides 1-2450 of SEQ.ID.No:9 (i.e., the entire β₂subunit coding sequence, plus 266 nucleotides of 5' untranslatedsequence and 675 nucleotides of 3' untranslated sequence) in operativeassociation with the T7 promoter. Therefore, Hβ2.1F was used forsynthesis of in vitro transcripts from the β₂ subunit DNA.

Since the 5' untranslated sequence of the β₂ subunit DNA contains apotential alternative translation initiation codon (ATG) beginning 11nucleotides upstream (nucleotides 256-258 in SEQ.ID.No:9) of the correcttranslation initiation codon (nucleotides 267-269 in SEQ.ID.No:9), andbecause the use of the upstream ATG sequence to initiate translation ofthe β₂ DNA would result in the generation of an inoperative peptide(because the upstream ATG is not in the correct reading frame), anadditional β₂ -encoding construct was prepared as follows. A 2.2 kbKspI/EcoRI DNA fragment containing nucleotides 262-2450 of SEQ.ID.No:9was ligated to pCMV-T7-2 in operative association with the T7 promoterof the plasmid to create pCMV-KEβ2. The β₂ subunit DNA contained inpCMV-KEβ2 retains only 5 nucleotides of 5' untranslated sequenceupstream of the correct translation initiation codon.

EXAMPLE 3 Expression of Recombinant Human Nicotinic AChR in Oocytes

Xenopus oocytes were injected with in vitro transcripts prepared fromconstructs containing DNA encoding α₃, α₇, β₂ and β₄ subunits.Electrophysiological measurements of the oocyte transmembrane currentswere made using the two-electrode voltage clamp technique (see, e.g.,Stuhmer (1992) Meth. Enzymol. 207:319-339).

1. Preparation of in vitro transcripts

Recombinant capped transcripts of pCMV-KEα3, pCMV-KEβ2, KEβ4.6/pGEM andpCMV-KEβ4 were synthesized from linearized plasmids using the mCAP RNACapping Kit (Cat. #200350 from Stratagene, Inc., La Jolla, Calif.).Recombinant capped transcripts of pCMV-KEα7, pCMV-KEα7.3 and Hβ2.1F weresynthesized from linearized plasmids using the MEGAscript T7 in vitrotranscription kit according to the capped transcript protocol providedby the manufacturer (Catalog #1334 from AMBION, Inc., Austin, Tex.). Themass of each synthesized transcript was determined by UV absorbance andthe integrity of each transcript was determined by electrophoresisthrough an agarose gel.

2. Electrophysiology

Xenopus oocytes were injected with either 12.5, 50 or 125 ng of humannicotinic AChR subunit transcript per oocyte. The preparation andinjection of oocytes were carried out as described by Dascal (1987) inCrit. Rev. Biochem. 22:317-387. Two-to-six days following mRNAinjection, the oocytes were examined using the two-electrode voltageclamp technique. The cells were bathed in Ringer's solution (115 mMNaCl, 2.5 mM KCl, 1.8 mM CaCl₂, 10 mM HEPES, pH 7.3) containing 1 μMatropine with or without 100 μM d-tubocurarine. Cells werevoltage-clamped at -60 to -80 mV. Data were acquired with Axotapesoftware at 2-5 Hz. The agonists acetylcholine (ACh), nicotine, andcytisine were added at concentrations ranging from 0.1 μM to 100 μM. Theresults of electrophysiological analyses of the oocytes are summarizedin Table 1.

                  TABLE 1                                                         ______________________________________                                                      Number of                                                       Template, ng RNA                                                                            oocytes              Current                                    injected      responding                                                                              Agonists   Amplitude                                  ______________________________________                                        pCMV-KEα3, 12.5 ng                                                                    0 of 8    ACh,                                                                          Nicotine                                              KEβ4.6/pGEM, 12.5 ng                                                                   0 of 9    ACh,                                                                          Nicotine                                              pCMV-KEα3, 12.5 ng                                                                    4 of 5    ACh,       20-550 nA                                  +                       Nicotine                                              KEβ4.6/pGEM, 12.5 ng                                                     pCMV-KEα3, 12.5 ng                                                                    3 of 4    ACh,       20-300 nA                                  +                       Cytisine,                                             KEβ4.6/pGEM, 12.5 ng                                                                             Nicotine                                              pCMV-KEα3, 125 ng                                                                     5 of 5    ACh,       200-500                                    +                       Nicotine,  nA                                         and pCMV-KEβ4, 125 ng                                                                            Cytisine                                              pCMV-KEα3, 125 ng                                                                     6 of 6    ACh,       100-400                                    +                       Nicotine,  nA                                         pCMV-KEβ4, 125 ng  Cytisine                                              pCMV-KEα7.3, 125 ng                                                                   3 of 15   ACh        ˜20 nA                               pCMV-KEα7, 125 ng                                                                     11 of 11  ACh        20-250 nA                                  pCMV-KEα3, 12.5 ng                                                                    2 of 9    ACh,       <10 nA                                     +                       Nicotine                                              pCMV-KEβ2, 12.5 ng                                                       pCMV-KEα3, 125 ng                                                                     0 of 9    ACh,                                                  +                       Nicotine                                              pCMV-KEβ2, 125 ng                                                        pCMV-KEα3, 125 ng                                                                     13 of 16  ACh (100 μM)                                                                          ˜20 nA                               +                       ACh (300 μM)                                                                          ˜80 nA                               Hβ2.1 F, 125 ng                                                          ______________________________________                                    

a. Oocytes Injected with α₃ and/or β₄ Transcripts

Oocytes that had been injected with 12.5 ng of the α₃ transcript or 12.5ng of the β₄ transcript did not respond to application of up to 100 μMACh, nicotine or cytisine. Thus, it appears that these subunits do notform functional homomeric nicotinic AChR channels. By contrast, oocytesinjected with 12.5 or 125 ng of the α₃ transcript and 12.5 ng or 125 ngof the β₄ transcript exhibited detectable inward currents in response toACh, nicotine, and cytisine at the tested concentrations (0.1 μM to 10μM). Some differences in the kinetics of the responses to cytisinecompared to nicotine and ACh were observed. The relative potency of theagonists appeared to be cytisine>ACh>nicotine, which differs from theresults of similar studies of oocytes injected with transcripts of therat nicotinic AChR α₃ and β₄ subunits (see, for example, Luetje et al.(1991) J. Neurosci. 11:837-845).

The responses to ACh and nicotine were reproducibly blocked byd-tubocurarine. For example, complete blockage of the response to AChwas observed in the presence of 100 μM d-tubocurarine. The inhibitionappeared to be reversible. The responses to ACh, nicotine and cytisinewere also at least partially blocked by 100 nM mecamylamine.

The current response of α₃ -β₄ -injected oocytes to 10 μM ACh was alsoexamined in terms of membrane voltage. In these experiments, voltagesteps were applied to the cells in the presence of ACh. The graph ofcurrent vs. voltage appeared typical of responses observed for Na⁺, K⁺-permeable channels. For example, the zero current level (reversalpotential) is less than -40 mV. The contribution of Ca⁺⁺ flux to thetotal current can be ascertained by varying the calcium concentration inthe external medium and taking multiple current measurements atdifferent holding potentials around the reversal potential. Such studiesindicate that the channel carrying the current generated in response toACh treatment of α₃ -β₄ -injected oocytes is permeable to Na⁺, K⁺ andCa⁺⁺.

b. Oocytes injected with a subunit transcripts

As described in Example 1, two constructs were prepared for use inexpressing the human neuronal nicotinic AChR α₇ subunit. PlasmidpCMV-KEα7.3 contains the α₇ subunit coding sequence with 72 nucleotidesof 5' untranslated sequence upstream of the translation initiationcodon. Plasmid pCMV-KEα7 contains the α₇ subunit coding sequence devoidof any 5' untranslated sequence and further contains a consensusribosome binding site immediately upstream of the coding sequence.

Oocytes injected with 125 ng of α₇ transcript synthesized from pCMV-KEα7displayed inward currents in response to 10 or 100 μM ACh. This responsewas blocked by 100 μM d-tubocurarine.

Oocytes injected with 125 ng of α₇ transcript synthesized frompCMV-KEα7.3 exhibited ACh-induced currents that were substantiallyweaker than those of oocytes injected with α₇ transcript synthesizedfrom pCMV-KEα7. These results indicate that human neuronal nicotinicAChR α₇ subunit transcripts generated from α₇ subunit DNA containing aribosome binding site in place of 5' untranslated sequence may bepreferable for expression of the α₇ receptor in oocytes.

c. Oocytes injected with α₃ and β₂ subunit transcripts

As described in Example 1, two constructs were prepared for use inexpressing the human neuronal nicotinic AChR β₂ subunit. Plasmid Hβ2.1Fcontains the β₂ subunit coding sequence with 266 nucleotides of 5'untranslated sequence upstream of the translation initiation codon.Plasmid pCMV-KEβ2 contains the β₂ subunit coding sequence and only 5nucleotides of 5' untranslated sequence upstream of the translationinitiation codon.

Oocytes injected with transcripts of pCMV-KEα3 and pCMV-KEβ2 displayedno current in response to nicotinic AChR α₃ agonists. In contrast,oocytes injected with transcripts of pCMV-KEα3 and Hβ2.1F displayed ˜20nA inward currents in response to 100 μM ACh and ˜80 nA inward currentsin response to 300 μM ACh. The current response was blocked by 100 μMd-tubocurarine. These results indicate that human neuronal nicotinicAChR β₂ subunit transcripts generated from β₂ subunit DNA containing 5'untranslated sequence may be preferable to transcripts generated from β₂DNA containing only a small portion of 5' untranslated sequence forexpression of the α₃ β₂ receptors in oocytes.

EXAMPLE 4 Recombinant Expression of Human nNAChR Subunits in MammalianCells

Human embryonic kidney (HEK) 293 cells were transiently and stablytransfected with DNA encoding human neuronal nicotinic AChR α₃ and β₄ ,or α₇ subunits. Transient transfectants were analyzed for expression ofnicotinic AChR using various assays, e.g., electrophysiological methods,Ca²⁺ -sensitive fluorescent indicator-based assays and ¹²⁵I!-α-bungarotoxin-binding assays.

1. Transient Transfection of HEK Cells

Two transient transfection were performed. In one transfection, HEKcells were transiently co-transfected with DNA encoding α₃ (plasmidpCMV-KEα3) and β₄ (plasmid pCMV-KFβ4) subunits. In the othertransfection, HEK cells were transiently transfected with DNA encodingthe α₇ subunit (plasmid pCMV-KEα7). In both transfections, ˜2×10⁶ HEKcells were transiently transfected with 18 μg of the indicatedplasmid(s) according to standard CaPO₄ transfection procedures Wigler etal. (1979) Proc. Natl. Acad. Sci. U.S.A. 76:1373-1376!. In addition, 2μg of plasmid pCMVβgal (Clontech Laboratories, Palo Alto, Calif.), whichcontains the Escherichia coli β-galactosidase gene fused to the CMVpromoter, were co-transfected as a reporter gene for monitoring theefficiency of transfection. The transfectants were analyzed forβ-galactosidase expression by measurement of β-galactosidase activityMiller (1972) Experiments in Molecular Genetics, pp.352-355, Cold SpringHarbor Press!. Transfectants can also be analyzed for β-galactosidaseexpression by direct staining of the product of a reaction involvingβ-galactosidase and the X-gal substrate Jones (1986) EMBO 5:3133-3142!.

The efficiency of transfection of HEK cells with pCMV-KEα3/pCMV-KEβ4 wastypical of standard efficiencies, whereas the efficiency of transfectionof HEK cells with pCMV-KEα7 was below standard levels.

2. Stable Transfection of HEK Cells

HEK cells were transfected using the calcium phosphate transfectionprocedure Current Protocols in Molecular Biology, Vol. 1, WileyInter-Science, Supplement 14, Unit 9.1.1-9.1.9 (1990)!. Ten-cm plates,each containing one-to-two million HEK cells were transfected with 1 mlof DNA/calcium phosphate precipitate containing 9.5 μg pCMV-KEα3, 9.5 μgpCMV-KEβ4 and 1 μg pSV2neo (as a selectable marker). After 14 days ofgrowth in media containing 1 μg/ml G418, colonies had formed and wereindividually isolated by using cloning cylinders. The isolates weresubjected to limiting dilution and screened to identify those thatexpressed the highest level of nicotinic AChR, as described below.

3. Analysis of Transfectants

a. Fluorescent indicator-based assays

Activation of the ligand-gated nicotinic AChR by agonists leads to aninflux of cations, including Ca⁺⁺, through the receptor channel. Ca⁺⁺entry into the cell through the channel can induce release of calciumcontained in intracellular stores. Monovalent cation entry into the cellthrough the channel can also result in an increase in cytoplasmic Ca⁺⁺levels through depolarization of the membrane and subsequent activationof voltage-dependent calcium channels. Therefore, methods of detectingtransient increases in intracellular calcium concentration can beapplied to the analysis of functional nicotinic AChR expression. Onemethod for measuring intracellular calcium levels relies oncalcium-sensitive fluorescent indicators.

Calcium-sensitive indicators, such as fluo-3 (Catalog No. F-1241,Molecular Probes, Inc., Eugene, Oreg.), are available as acetoxymethylesters which are membrane permeable. When the acetoxymethyl ester formof the indicator enters a cell, the ester group is removed by cytosolicesterases, thereby trapping the free indicator in the cytosol.Interaction of the free indicator with calcium results in increasedfluorescence of the indicator; therefore, an increase in theintracellular Ca²⁺ concentration of cells containing the indicator canbe expressed directly as an increase in fluorescence. An automatedfluorescence detection system for assaying icotinic AChR has beendescribed in commonly assigned pending U.S. Pat. application Ser. No.07/812,254 and corresponding PCT Patent Application No. US92/11090.

HEK cells that were transiently or stably co-transfected with DNAencoding α3 and β4 subunits were analyzed for expression of functionalrecombinant nicotinic AChR using the automated fluorescentindicator-based assay. The assay procedure was as follows.

Untransfected HEK cells (or HEK cells transfected with pCMV-T7-2) andHEK cells that had been co-transfected with pCMV-KEα3 and pCMV-KEβ4 wereplated in the wells of a 96-well microtiter dish and loaded with fluo-3by incubation for 2 hours at 20° C. in a medium containing 20 μM fluo-3,0.2% Pluronic F-127 in HBS (125 mM NaCl, 5 mM KCl, 1.8 mM CaCl₂, 0.62 mMMgSO₄, 6 mM glucose, 20 mM HEPES, pH 7.4). The cells were then washedwith assay buffer (i.e., HBS). The antagonist d-tubocurarine was addedto some of the wells at a final concentration of 10 μM. The microtiterdish was then placed into a fluorescence plate reader and the basalfluorescence of each well was measured and recorded before addition of200 μM nicotine to the wells. The fluorescence of the wells wasmonitored repeatedly during a period of approximately 60 secondsfollowing addition of nicotine.

The fluorescence of the untransfected HEK cells (or HEK cellstransfected with pCMV-T7-2) did not change after addition of nicotine.In contrast, the fluorescence of the co-transfected cells, in theabsence of d-tubocurarine, increased dramatically after addition ofnicotine to the wells. This nicotine-stimulated increase in fluorescencewas not observed in co-transfected cells that had been exposed to theantagonist d-tubocurarine. These results demonstrate that theco-transfected cells express functional recombinant AChR that areactivated by nicotine and blocked by d-tubocurarine.

b. α-Bungarotoxin binding assays

HEK293 cells transiently transfected with pCMV-KEα7 were analyzed for¹²⁵ I!-α-bungarotoxin (BgTx) binding. Both whole transfected cells andmembranes prepared from transfected cells were examined in these assays.Rat brain membranes were included in the assays as a positive control.

Rat brain membranes were prepared according to the method of Hampson etal. (1987) J. Neurochem 49:1209. Membranes were prepared from the HEKcells transfected with pCMV-KEα7 and HEK cells transiently transfectedwith plasmid pUC19 only (negative control) according to the method ofPerez-Reyes et al. (1989) Nature 340:233. Whole transfected and negativecontrol cells were obtained by spraying the tissue culture plates withphosphate-buffered saline containing 0.1% (w/v) BSA. The cells were thencentrifuged at low speed, washed once, resuspended in assay buffer (118mM NaCl, 4.8 mM KCl, 2.5 mM CaCI₂, 1.2 mM MgSO₄, 20 mM HEPES, 0.1%(w/v)BSA, 0.05% (w/v) bacitracin and 0.5 mM PMSF, pH 7.5) and counted.

Specific binding of ¹²⁵ I!-α-BgTx to rat brain membranes was determinedessentially as described by Marks et al. (1982) Molec. Pharmacol.22:554-564, with several modifications. The membranes were washed twicein assay buffer. The assay was carried out in 12×75 mm polypropylenetest tubes in a total volume of 0.5 ml assay buffer. The membranes wereincubated with 10 nM ¹²⁵ I!-α-BgTx (New England Nuclear, Boston, Mass.)for one hour at 37° C. The assay mixtures were then centrifuged at2300×g for 10 minutes at 4° C. The supernatant was decanted and thepellets were washed twice with 2 ml aliquots of ice-cold assay buffer.The supernatants were decanted again and the radioactivity of thepellets was measured in a γ-counter. Non-specific binding was determinedin the presence of 1 μM unlabeled α-BgTx. Specific binding wasdetermined by subtracting nonspecific binding from total binding.Specific binding of ¹²⁵ I!-α-BgTx to membranes prepared from transfectedand negative control cells was determined as described for determiningspecific binding to rat brain membranes except that the assay buffer didnot contain BSA, bacitracin and PMSF. Specific binding of ¹²⁵ I!-α-BgTxto transfected and negative control whole cells was determined basicallyas described for determining specific binding to rat brain membranes.

¹²⁵ I!-α-BgTx binding was evaluated as a function of membraneconcentration and as a function of incubation time. ¹²⁵ I!-α-BgTxbinding to rat brain membranes increased in a linear fashion withincreasing amounts of membrane (ranging between 25-500 μg). The overallsignal-to-noise ratio of binding (i.e., ratio of total binding tonon-specific binding) was 3:1. Although some binding of ¹²⁵ I!-α-BgTx totransfected cell membranes was detected, it was mostly non-specificbinding and did not increase with increasing amounts of membrane. ¹²⁵I!-α-BgTx binding to the transfectants and negative control cellsappeared to be similar.

To monitor ¹²⁵ I!-α-BgTx binding to rat brain membranes and wholetransfected and negative control cells, 300 μg of membrane or 500,000cells were incubated with 1 nM or 10 nM ¹²⁵ I!-α-BgTx, respectively, at37° C. for various times ranging from 0-350 min. Aliquots of assaymixture were transferred to 1.5 ml microfuge tubes at various times andcentrifuged. The pellets were washed twice with assay buffer. ¹²⁵I!-α-BgTx binding to rat brain membranes increased with time and wasmaximal after three hours. The binding profiles of the transfected andnegative control cells were the same and differed from that of rat brainmembranes.

EXAMPLE 5 Characterization of Cell Lines Expressing nNAChRs

Recombinant cell lines generated by transfection with DNA encoding humanneuronal nicotinic AChRs, such as those described in Example 3, can befurther characterized using one or more of the following methods.

A. Northern or slot blot analysis for expression of α- and/or β-subunitencoding messages

Total RNA is isolated from ˜1×10⁷ cells and 10-15 μg of RNA from eachcell type is used for northern or slot blot hybridization analysis. Theinserts from human neuronal NAChR-encoding plasmids can benick-translated and used as probe. In addition, the β-actin genesequence (Cleveland et al. (1980) Cell 20:95-105) can be nick-translatedand used as a control probe on duplicate filters to confirm the presenceor absence of RNA on each blot and to provide a rough standard for usein quantitating differences in α- or β-specific mRNA levels between celllines. Typical northern and slot blot hybridization and wash conditionsare as follows:

hybridization in 5× SSPE, 5× Denhardt's solution, 50% formamide, at 42°C. followed by washing in 0.2× SSPE, 0.1% SDS, at 65° C.

B. Nicotine-binding assay

Cell lines generated by transfection with human neuronal nicotinic AChRα- or α- and β-subunit-encoding DNA can be analyzed for their ability tobind nicotine, for example, as compared to control cell lines:neuronally-derived cell lines PC12 (Boulter et al., (1986), supra; ATCC#CRL1721) and IMR32 (Clementi, et al. (1986); Int. J. Neurochem.47:291-297; ATCC #CCL127), and muscle-derived cell line BC3H1 (Patrick,et al., (1977); J. Biol. Chem. 252:2143-2153). Negative control cells(i.e., host cells from which the transfectants were prepared) are alsoincluded in the assay. The assay is conducted as follows:

Just prior to being assayed, transfected cells are removed from platesby scraping. Positive control cells used are PC12, BC3H1, and IMR32(which had been starved for fresh media for seven days). Control celllines are removed by rinsing in 37° C. assay buffer (50 mM Tris/HCl, 1mM MgCl₂, 2 mM CaCl₂, 120 mM NaCl, 3 mM EDTA, 2 mg/ml BSA and 0.1%aprotinin at pH7.4). The cells are washed and resuspended to aconcentration of 1×10⁶ /250 μl. To each plastic assay tube is added 250μl of the cell solution, 15 nM ³ H-nicotine, with or without 1 mMunlabeled nicotine, and assay buffer to make a final volume of 500 μl.The assays for the transfected cell lines are incubated for 30 min atroom temperature; the assays of the positive control cells are incubatedfor 2 min at 1° C. After the appropriate incubation time, 450 μlaliquots of assay volume are filtered through Whatman GF/C glass fiberfilters which has been pretreated by incubation in 0.05%polyethyleneimine for 24 hours at 4° C. The filters are then washedtwice, with 4 ml each wash, with ice cold assay buffer. After washing,the filters are dried, added to vials containing 5 ml scintillationfluid and radioactivity is measured.

C. ⁸⁶ Rb ion-flux assay

The ability of nicotine or nicotine agonists and antagonists to mediatethe influx of ⁸⁶ Rb into transfected and control cells has been found toprovide an indication of the presence of functional AChRs on the cellsurface. The ⁸⁶ Rb ion-flux assay is conducted as follows:

1. The night before the experiment, cells are plated at 2×10⁶ per well(i.e., 2 ml per well) in a 6-well polylysine-coated plate.

2. The culture medium is decanted and the plate washed with 2 ml ofassay buffer (50 mM HEPES, 260 mM sucrose, 5.4 mM KCl, 1.8 mM CaCl₂, 0.8mM MgsO₄, 5.5. mM glucose) at room temperature.

3. The assay buffer is decanted and 1 ml of assay buffer, containing 3μCi/ml ⁸⁶ Rb, with 5 mM ouabain and agonist or antagonist in aconcentration to effect a maximum response, is added.

4. The plate is incubated on ice at 1° C. for 4 min.

5. The buffer is decanted into a waste container and each well waswashed with 3 ml of assay buffer, followed by two washes of 2 ml each.

6. The cells are lysed with 2×0.5 ml of 0.2% SDS per well andtransferred to a scintillation vial containing 5 ml of scintillationfluid.

7. The radioactivity contained in each vial is measured and the datacalculated.

Positive control cells provided the following data in this assay:

    ______________________________________                                                 PC12         IMR32                                                                   Maximum            Maximum                                             EC.sub.50                                                                            response  EC.sub.50                                                                              response                                   ______________________________________                                        Agonist                                                                       nicotine     52 μM                                                                             2.1 X.sup.a                                                                             18 μM                                                                             7.7 X.sup.a                              CCh*         35 μM                                                                             3.3 X.sup.b                                                                             230 μM                                                                            7.6 X.sup.c                              cytisine     57 μM                                                                             3.6 X.sup.d                                                                             14 μM                                                                              10 X.sup.e                              Antagonist                                                                    d-tubocurarine                                                                           0.81 μM         2.5 μM                                       mecamylamine                                                                             0.42 μM         0.11 μM                                      hexamethonium                                                                            nd.sup.f           22 μM                                        atropine   12.5 μM         43 μM                                        ______________________________________                                         *CCh = carbamylcholine                                                        .sup.a 200 μM nicotine                                                     .sup.b 300 μM CCh                                                          .sup.c 3 mM CCh                                                               .sup.d 1 mM cytisine                                                          .sup.e 100 μM cytisine                                                     .sup.f nd = not determined                                               

D. Electrophysiological Analysis of Mammalian Cells Transfected withHuman Neuronal Nicotinic AChR Subunit-encoding DNA

Electrophysiological measurements may be used to assess the activity ofrecombinant receptors or to assess the ability of a test compound topotentiate, antagonize or otherwise modulate the magnitude and durationof the flow of cations through the ligand-gated recombinant AChR. Thefunction of the expressed neuronal AChR can be assessed by a variety ofelectrophysiological techniques, including two-electrode voltage clampand patch clamp methods. The cation-conducting channel intrinsic to theAChR opens in response to acetylcholine (ACh) or other nicotiniccholinergic agonists, permitting the flow of transmembrane currentcarried predominantly by sodium and potassium ions under physiologicalconditions. This current can be monitored directly by voltage clamptechniques. In preferred embodiments, transfected mammalian cells orinjected oocytes are analyzed electrophysiologically for the presence ofAChR agonist-dependent currents.

While the invention has been described in detail with reference tocertain preferred embodiments thereof, it will be understood thatmodifications and variations are within the spirit and scope of thatwhich is described and claimed.

Summary of Sequences

Sequence ID No. 1 is a nucleotide sequence encoding an α₂ subunit ofhuman neuronal nicotinic acetylcholine receptor, and the deduced aminoacid sequence thereof.

Sequence ID No. 2 is the amino acid sequence of the α₂ subunit of humanneuronal nicotinic acetylcholine receptor set forth in Sequence ID No.1.

Sequence ID No. 3 is a nucleotide sequence encoding an α₃ subunit ofhuman neuronal nicotinic acetylcholine receptor, and the deduced aminoacid sequence thereof.

Sequence ID No. 4 is the amino acid sequence of the α₃ subunit of humanneuronal nicotinic acetylcholine receptor set forth in Sequence ID No.3.

Sequence ID No. 5 is a nucleotide sequence encoding an α₄ subunit of ahuman neuronal nicotinic acetylcholine receptor, and the deduced aminoacid sequence thereof.

Sequence ID No. 6 is the amino acid sequence of the α₄ subunit of ahuman neuronal nicotinic acetylcholine receptor set forth in Sequence IDNo. 5.

Sequence ID No. 7 is a nucleotide sequence encoding an α₇ subunit ofhuman neuronal nicotinic acetylcholine receptor, and the deduced aminoacid sequence thereof.

Sequence ID No. 8 is the amino acid sequence of the α₇ subunit of humanneuronal nicotinic acetylcholine receptor set forth in Sequence ID No.7.

Sequence ID No. 9 is a nucleotide sequence encoding a β₂ subunit ofhuman neuronal nicotinic acetylcholine receptor, and the deduced aminoacid sequence thereof.

Sequence ID No. 10 is the amino acid sequence of the β₂ subunit of humanneuronal nicotinic acetylcholine receptor set forth in Sequence ID No.9.

Sequence ID No. 11 is a nucleotide sequence encoding a β₄ subunit ofhuman neuronal nicotinic acetylcholine receptor, and the deduced aminoacid sequence thereof.

Sequence ID No. 12 is the amino acid sequence of the β₄ subunit of humanneuronal nicotinic acetylcholine receptor set forth in Sequence ID No.11.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 12                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2068 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: both                                                            (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 166..1752                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CAATGACCTGTTTTCTTCTGTAACCACAGGTTCGGTGGTGAGAGGAACCTTCGCAGAATC60                CAGCAGAATCCTCACAGAATCCAGCAGCAGCTCTGCTGGGGACATGGTCCATGGTGCAAC120               CCACAGCAAAGCCCTGACCTGACCTCCTGATGCTCAGGAGAAGCCATGGGCCCCTCCTGT180               CCTGTGTTCCTGTCCTTCACAAAGCTCAGCCTGTGGTGGCTCCTTCTGACCCCAGCAGGT240               GGAGAGGAAGCTAAGCGCCCACCTCCCAGGGCTCCTGGAGACCCACTCTCCTCTCCCAGT300               CCCACGGCATTGCCGCAGGGAGGCTCGCATACCGAGACTGAGGACCGGCTCTTCAAACAC360               CTCTTCCGGGGCTACAACCGCTGGGCGCGCCCGGTGCCCAACACTTCAGACGTGGTGATT420               GTGCGCTTTGGACTGTCCATCGCTCAGCTCATCGATGTGGATGAGAAGAACCAAATGATG480               ACCACCAACGTCTGGCTAAAACAGGAGTGGAGCGACTACAAACTGCGCTGGAACCCCGCT540               GATTTTGGCAACATCACATCTCTCAGGGTCCCTTCTGAGATGATCTGGATCCCCGACATT600               GTTCTCTACAACAAANNTGGGGAGTTTGCAGTGACCCACATGACCAAGGCCCACCTCTTC660               TCCACGGGCACTGTGCACTGGGTGCCCCCGGCCATCTACAAGAGCTCCTGCAGCATCGAC720               GTCACCTTCTTCCCCTTCGACCAGCAGAACTGCAAGATGAAGTTTGGCTCCTGGACTTAT780               GACAAGGCCAAGATCGACCTGGAGCAGATGGAGCAGACTGTGGACCTGAAGGACTACTGG840               GAGAGCGGCGAGTGGGCCATCGTCAATGCCACGGGCACCTACAACAGCAAGAAGTACGAC900               TGCTGCGCCGAGATCTACCCCGACGTCACCTACGCCTTCGTCATCCGGCGGCTGCCGCTC960               TTCTACACCATCAACCTCATCATCCCCTGCCTGCTCATCTCCTGCCTCACTGTGCTGGTC1020              TTCTACCTGCCCTCCGACTGCGGCGAGAAGATCACGCTGTGCATTTCGGTGCTGCTGTCA1080              CTCACCGTCTTCCTGCTGCTCATCACTGAGATCATCCCGTCCACCTCGCTGGTCATCCCG1140              CTCATCGGCGAGTACCTGCTGTTCACCATGATCTTCGTCACCCTGTCCATCGTCATCACC1200              GTCTTCGTGCTCAATGTGGACCACCGCTCCCCCAGCACCCACACCATGCCCCACTGGGTG1260              CGGGGGGCCCTTCTGGGCTGTGTGCCCCGGTGGCTTCTGATGAACCGGCCCCCACCACCC1320              GTGGAGCTCTGCCACCCCCTACGCCTGAAGCTCAGCCCCTCTTATCACTGGCTGGAGAGC1380              AACGTGGATGCCGAGGAGAGGGAGGTGGTGGTGGAGGAGGAGGACAGATGGGCATGTGCA1440              GGTCATGTGGCCCCCTCTGTGGGCACCCTCTGCAGCCACGGCCACCTGCACTCTGGGGCC1500              TCAGGTCCCAAGGCTGAGGCTCTGCTGCAGGAGGGTGAGCTGCTGCTATCACCCCACATG1560              CAGAAGGCACTGGAAGGTGTGCACTACATTGCCGACCACCTGCGGTCTGAGGATGCTGAC1620              TCTTCGGTGAAGGAGGACTGGAAGTATGTTGCCATGGTCATCGACAGGATCTTCCTCTGG1680              CTGTTTATCATCGTCTGCTTCCTGGGGACCATCGGCCTCTTTCTGCCTCCGTTCCTAGCT1740              GGAATGATCTGACTGCACCTCCCTCGAGCTGGCTCCCAGGGCAAAGGGGAGGGTTCTTGG1800              ATGTGGAAGGGCTTTGAACAATGTTTAGATTTGGAGATGAGCCCAAAGTGCCAGGGAGAA1860              CAGCCAGGTGAGGTGGGAGGTTGGAGAGCCAGGTGAGGTCTCTCTAAGTCAGGCTGGGGT1920              TGAAGTTTGGAGTCTGTCCGAGTTTGCAGGGTGCTGAGCTGTATGGTCCAGCAGGGGAGT1980              AATAAGGGCTCTTCCCGAAGGGGAGGAAGCGGGAGGCAGCGCCTGCACCTGATGTGGAGG2040              TACAGAGCAGATCTTCCCTACCGGGGAG2068                                              (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 528 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetGlyProSerCysProValPheLeuSerPheThrLysLeuSerLeu                              151015                                                                        TrpTrpLeuLeuLeuThrProAlaGlyGlyGluGluAlaLysArgPro                              202530                                                                        ProProArgAlaProGlyAspProLeuSerSerProSerProThrAla                              354045                                                                        LeuProGlnGlyGlySerHisThrGluThrGluAspArgLeuPheLys                              505560                                                                        HisLeuPheArgGlyTyrAsnArgTrpAlaArgProValProAsnThr                              65707580                                                                      SerAspValValIleValArgPheGlyLeuSerIleAlaGlnLeuIle                              859095                                                                        AspValAspGluLysAsnGlnMetMetThrThrAsnValTrpLeuLys                              100105110                                                                     GlnGluTrpSerAspTyrLysLeuArgTrpAsnProAlaAspPheGly                              115120125                                                                     AsnIleThrSerLeuArgValProSerGluMetIleTrpIleProAsp                              130135140                                                                     IleValLeuTyrAsnLysXaaGlyGluPheAlaValThrHisMetThr                              145150155160                                                                  LysAlaHisLeuPheSerThrGlyThrValHisTrpValProProAla                              165170175                                                                     IleTyrLysSerSerCysSerIleAspValThrPhePheProPheAsp                              180185190                                                                     GlnGlnAsnCysLysMetLysPheGlySerTrpThrTyrAspLysAla                              195200205                                                                     LysIleAspLeuGluGlnMetGluGlnThrValAspLeuLysAspTyr                              210215220                                                                     TrpGluSerGlyGluTrpAlaIleValAsnAlaThrGlyThrTyrAsn                              225230235240                                                                  SerLysLysTyrAspCysCysAlaGluIleTyrProAspValThrTyr                              245250255                                                                     AlaPheValIleArgArgLeuProLeuPheTyrThrIleAsnLeuIle                              260265270                                                                     IleProCysLeuLeuIleSerCysLeuThrValLeuValPheTyrLeu                              275280285                                                                     ProSerAspCysGlyGluLysIleThrLeuCysIleSerValLeuLeu                              290295300                                                                     SerLeuThrValPheLeuLeuLeuIleThrGluIleIleProSerThr                              305310315320                                                                  SerLeuValIleProLeuIleGlyGluTyrLeuLeuPheThrMetIle                              325330335                                                                     PheValThrLeuSerIleValIleThrValPheValLeuAsnValAsp                              340345350                                                                     HisArgSerProSerThrHisThrMetProHisTrpValArgGlyAla                              355360365                                                                     LeuLeuGlyCysValProArgTrpLeuLeuMetAsnArgProProPro                              370375380                                                                     ProValGluLeuCysHisProLeuArgLeuLysLeuSerProSerTyr                              385390395400                                                                  HisTrpLeuGluSerAsnValAspAlaGluGluArgGluValValVal                              405410415                                                                     GluGluGluAspArgTrpAlaCysAlaGlyHisValAlaProSerVal                              420425430                                                                     GlyThrLeuCysSerHisGlyHisLeuHisSerGlyAlaSerGlyPro                              435440445                                                                     LysAlaGluAlaLeuLeuGlnGluGlyGluLeuLeuLeuSerProHis                              450455460                                                                     MetGlnLysAlaLeuGluGlyValHisTyrIleAlaAspHisLeuArg                              465470475480                                                                  SerGluAspAlaAspSerSerValLysGluAspTrpLysTyrValAla                              485490495                                                                     MetValIleAspArgIlePheLeuTrpLeuPheIleIleValCysPhe                              500505510                                                                     LeuGlyThrIleGlyLeuPheLeuProProPheLeuAlaGlyMetIle                              515520525                                                                     (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1756 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: both                                                            (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 39..1553                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CCGACCGTCCGGGTCCGCGGCCAGCCCGGCCACCAGCCATGGGCTCTGGCCCGCTCTCGC60                TGCCCCTGGCGCTGTCGCCGCCGCGGCTGCTGCTGCTGCTGCTGTCTCTGCTGCCAGTGG120               CCAGGGCCTCAGAGGCTGAGCACCGTCTATTTGAGCGGCTGTTTGAAGATTACAATGAGA180               TCATCCGGCCTGTGGCCAACGTGTCTGACCCAGTCATCATCCATTTCGAGGTGTCCATGT240               CTCAGCTGGTGAAGGTGGATGAAGTAAACCAGATCATGGAGACCAACCTGTGGCTCAAGC300               AAATCTGGMATGACTACAAGCTGAAGTGGAACCCCTCTGACTATGGTGGGGCAGAGTTCA360               TGCGTGTCCCTGCACAGAAGATCTGGAAGCCAGACATTGTGCTGTATAACAATGCTGTTG420               GGGATTTCCAGGTGGACGACAAGACCAAAGCCTTACTCAAGTACACTGGGGAGGTGACTT480               GGATACCTCCGGCCATCTTTAAGAGCTCCTGTAAAATCGACGTGACCTACTTCCCGTTTG540               ATTACCAAAACTGTACCATGAAGTTCGGTTCCTGGTCCTACGATAAGGCGAAAATCGACC600               TGGTCCTGATCGGCTCTTCCATGAACCTCAAGGACTATTGGGAGAGCGGCGAGTGGGCCA660               TCATCAAAGCCCCAGGYTATAACCACGACATCAAGTACAACTGCTGCGAGGAGATCTACC720               CCGACATCACATACTCGCTGATCATCCGGCGGCTGTCGTTGTTCTACACCATCATCCTCA780               TCATCCCCTGGCTGATCATCTCCTTCATCACTGTGGTCGTCTTCTACCTGCCCTCCGACT840               GCGGTGAGAAGGTGACCCTGTGCATTTCTGTCCTCCTCTCCCTGACGGTGTTTCTCCTGG900               TGATCACTGAGACCATCCCTTCCACCTCGCTGGTCATCCCCCTGATTGGAGAGTACCTCC960               TGTTCACCATGATTTTTGTAACCTTGTCCATCGCCATCACCGTCTTCGTGCTCAACGTGC1020              ACTACAGAACCCCGACGACACACACAATGCCCTCATGGGTGAAGACTGTATTCTTGAACC1080              TGCTCCCCAGGGTCATGTTCATGACCAGGCCAACAAGCAACGAGGGCAACGCTCAGAAGC1140              CGAGGCCCCTCTACGGTGCCGAGCTCTCAAATCTGAATTGCTTCAGCCGCGCAGAGTCCA1200              WAGGCTGCAAGGAGGGCTACCCCTGCCAGGACGGGATGTGTGGTTACTGCCACCACCGCA1260              GGATAAAAATCTCCAATTTCAGTGCTAACCTCACGAGAAGCTCTAGTTCTGAATCTGTTG1320              ATGCTGTGCTGTCCCTCTCTGCTTTGTCACCAGAAATCAAAGAAGCCATCCAAAGTGTCA1380              AGTATATTGCTGAAAATATGAAAGCACAAAATGAAGCCAAAGAGATTCAAGATGATTGGA1440              AGTATGTTGCCATGGTGATTGATCGCATTTTTCTGTGGGTTTTCACCCTGGTGTGCATTC1500              TAGGGACAGAAGGATTGTTTCTGCAACCCCTGATGGCCAGGGAAGATGCATAAGCACTAA1560              GCTGTGTGTCTGTCTGGGAGAGTTCCCTGTGTCAGAGAAGAGGGAGGCTGCTTCCTAGTA1620              AGAACGTACTTTCTGTTATCAAGCTACCAGCTTTGTTTTTGGCATTTCGAGGTTTACTTA1680              TTTTCCACTTATCTTGGAATCATGCGCGAMMAAATGTCAAGAGTATTTATTACCGATAAA1740              TGAACATTTAACTAGC1756                                                          (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 504 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetGlySerGlyProLeuSerLeuProLeuAlaLeuSerProProArg                              151015                                                                        LeuLeuLeuLeuLeuLeuSerLeuLeuProValAlaArgAlaSerGlu                              202530                                                                        AlaGluHisArgLeuPheGluArgLeuPheGluAspTyrAsnGluIle                              354045                                                                        IleArgProValAlaAsnValSerAspProValIleIleHisPheGlu                              505560                                                                        ValSerMetSerGlnLeuValLysValAspGluValAsnGlnIleMet                              65707580                                                                      GluThrAsnLeuTrpLeuLysGlnIleTrpXaaAspTyrLysLeuLys                              859095                                                                        TrpAsnProSerAspTyrGlyGlyAlaGluPheMetArgValProAla                              100105110                                                                     GlnLysIleTrpLysProAspIleValLeuTyrAsnAsnAlaValGly                              115120125                                                                     AspPheGlnValAspAspLysThrLysAlaLeuLeuLysTyrThrGly                              130135140                                                                     GluValThrTrpIleProProAlaIlePheLysSerSerCysLysIle                              145150155160                                                                  AspValThrTyrPheProPheAspTyrGlnAsnCysThrMetLysPhe                              165170175                                                                     GlySerTrpSerTyrAspLysAlaLysIleAspLeuValLeuIleGly                              180185190                                                                     SerSerMetAsnLeuLysAspTyrTrpGluSerGlyGluTrpAlaIle                              195200205                                                                     IleLysAlaProGlyTyrAsnHisAspIleLysTyrAsnCysCysGlu                              210215220                                                                     GluIleTyrProAspIleThrTyrSerLeuIleIleArgArgLeuSer                              225230235240                                                                  LeuPheTyrThrIleIleLeuIleIleProTrpLeuIleIleSerPhe                              245250255                                                                     IleThrValValValPheTyrLeuProSerAspCysGlyGluLysVal                              260265270                                                                     ThrLeuCysIleSerValLeuLeuSerLeuThrValPheLeuLeuVal                              275280285                                                                     IleThrGluThrIleProSerThrSerLeuValIleProLeuIleGly                              290295300                                                                     GluTyrLeuLeuPheThrMetIlePheValThrLeuSerIleAlaIle                              305310315320                                                                  ThrValPheValLeuAsnValHisTyrArgThrProThrThrHisThr                              325330335                                                                     MetProSerTrpValLysThrValPheLeuAsnLeuLeuProArgVal                              340345350                                                                     MetPheMetThrArgProThrSerAsnGluGlyAsnAlaGlnLysPro                              355360365                                                                     ArgProLeuTyrGlyAlaGluLeuSerAsnLeuAsnCysPheSerArg                              370375380                                                                     AlaGluSerXaaGlyCysLysGluGlyTyrProCysGlnAspGlyMet                              385390395400                                                                  CysGlyTyrCysHisHisArgArgIleLysIleSerAsnPheSerAla                              405410415                                                                     AsnLeuThrArgSerSerSerSerGluSerValAspAlaValLeuSer                              420425430                                                                     LeuSerAlaLeuSerProGluIleLysGluAlaIleGlnSerValLys                              435440445                                                                     TyrIleAlaGluAsnMetLysAlaGlnAsnGluAlaLysGluIleGln                              450455460                                                                     AspAspTrpLysTyrValAlaMetValIleAspArgIlePheLeuTrp                              465470475480                                                                  ValPheThrLeuValCysThrLeuGlyThrGluGlyLeuPheLeuGln                              485490495                                                                     ProLeuMetAlaArgGluAspAla                                                      500                                                                           (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2374 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: both                                                            (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 184..2067                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       TCCACAAGCCGGCGCTCGCTGCGGCGCCGCCGCCGCGCCGCGCGCCACAGGAGAAGGCGA60                GCCGGGCCCGGCGGCCGAAGCGGCCCGCGAGGCGCGGGAGGCATGAAGTTGGGCGCGCAC120               GGGCCTCGAAGCGGCGGGGAGCCGGGAGCCGCCCGCATCTAGAGCCCGCGAGGTGCGTGC180               GCCATGGAGCTAGGGGGCCCCGGAGCGCCGCGGCTGCTGCCGCCGCTGCTGCTGCTTCTG240               GGGACCGGCCTCCTGCGCGCCAGCAGCCATGTGGAGACCCGGGCCCACGCCGAGGAGCGG300               CTCCTGAAGAAACTCTTCTCCGGTTACAACAAGTGGTCCCGACCCGTGGCCAACATCTCG360               GACGTGGTCCTCGTCCGCTTCGGCCTGTCCATCGCTCAGCTCATTGACGTGGATGAGAAG420               AACCAGATGATGACCACGAACGTCTGGGTGAAGCAGGAGTGGCACGACTACAAGCTGCGC480               TGGGACCCAGCTGACTATGAGAATGTCACCTCCATCCGCATCCCCTCCGAGCTCATCTGG540               CGGCCGGACATCGCCCTCTACAACAATGCTGACGGGGACTTCGCGGCCACCCACCTGACC600               AAGGCCCACCTGTTCCATGACGGGCGGGTGCAGCGGACTCCCCCGGCCATTTACAAGAGC660               TCCTGCAGCATCGACGTCACCTTCTTCCCCTTCGACCAGCAGAACTGCACCATGAAATTC720               GGCTCCTGGACCTACGACAAGGCCAAGATCGACCTGGTGAACATGCACAGCCGCGTGGAC780               CAGCTGGACTTCTGGGAGAGTGGCGAGTGGCTCATCGCGGACGCCGYGGGCACCTACAAC840               ACCAGGAAGTACGAGTGCTGCGCCGAGATCTACCCGGACATCACCTATGCCTACGCCATC900               CGGCGGCTGCCGCTCTTCTGCACCATCAACCTCATCATCCCCTGGCTGCTCATCTCCTGC960               CTCACCGCGCTGGTCTTCTACCTGCCCTCCGAGTGTGGCGAGAAGATCACGCTGTGCATC1020              TCCGCGCTGCTGTCGCTCACCGGCTTCCTGCTGCTCATCACCGAGATCATCCCGCCCACC1080              TCACTGGTCATCCCACTCATCGGCGAGTACCTGCTGTTCACCATGATCTTCGTCACCCTG1140              TCCATCGCCATCACGGTCTTCGTGCTCAACGTGCACCACCGCTCGCCACGCACGCACACC1200              ATGCCCACCTGGGTACGCAGCGTCTTCCTGGACATCGTGCCACGCCTGCTCCTCATGAAG1260              CGGCCGTCCGTGGTCAAGGACAATTGCCGGCGGCTCATCGAGTCCATGCATAAGATGGCC1320              AGTGCCCCGCGCTTCTGGCCCGAGCCAGAAGGGGAGCCCCCTGCCACGAGCGGCACCCAG1380              AGCCTGCACCCTCCCTCACCGCCCTTCTGCGTCCCCCTGGATGTGCCGGCTGAGCCTGGG1440              CCTTCCTGCAAGTCACCCTCCGACCAGCTCCCTCCTCAGAAGCCCCTGGAAGCTGAGAAA1500              GACAGCCCCCACCCCTCGCCTGGACCCTGCCGCCCGCCCCACGGCACCCAGGCACCAGGG1560              CTGGCCAAAGCCAGGTCCCTCAGCGTCCAGCACATGTCCAGCCCTGGCGAAGCGGTGGAA1620              GGCGGCGTCCGGTGCCGGTCTCGGAGCATCCAGTACTGTGTTCCCCGAGACGATGCCGCC1680              CCCGAGGCAGATGGCCAGGCTKCCGGCGCCCTGGCCTCTCGCAACAGCCACTCGGCTGAG1740              CTCCCACCCCCAGACCAGCCCTCTCCGTGCAAATGCACATGCAAGAAGGAGCCCTCTTCG1800              GTGTCCCCGAGCGCCRCGGTCAAGACCCGCAGCACCAAAGCGCCGCCGCCGCACCTGCCC1860              CTGTCGCCGGCCCTGAGCCGGGCGGTGGAGGGCGTCCAGTACATTGCAGACCACCTGAAG1920              GCCGAAGACACAGACTTCTCGGTGAAGGAGGACTGGAAGTACGTGGCCATGGTCATCGAC1980              CGCATCTTCCTCTGGATGTTCATCATCGTCTGCCTGCTGGGGACGGTGGGCCTCTTCCTG2040              CCGCCCTGGCTGGCTGGCATGATCTAGGAAGGGACCGGGAGCCTGCGTGGCCTGGGGCTG2100              CCGCGCACGGGGCCAGCATCCATGCGGCCGGCCTGGGGCCGGGCTGGCTTCTCCCTGGAC2160              TCTGTGGGGCCACACGTTTGCCAAATTTCCCTYCCTGTTCTGTGTCTGCTGTAAGACGGC2220              CTTGGACGGCGACACGGCCTCTGGGGAGACCGAGTGTGGAGCTGCTTCCAGTTGGACTCT2280              SGCCTCAGNAGGCAGCGGCTTGGAGCAGAGGTGGCGGTCGCCGCCTYCTACCTGCAGGAC2340              TCGGGCTAAGTCCAGCTCTCCCCCTGCGCAGCCC2374                                        (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 627 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       MetGluLeuGlyGlyProGlyAlaProArgLeuLeuProProLeuLeu                              151015                                                                        LeuLeuLeuGlyThrGlyLeuLeuArgAlaSerSerHisValGluThr                              202530                                                                        ArgAlaHisAlaGluGluArgLeuLeuLysLysLeuPheSerGlyTyr                              354045                                                                        AsnLysTrpSerArgProValAlaAsnIleSerAspValValLeuVal                              505560                                                                        ArgPheGlyLeuSerIleAlaGlnLeuIleAspValAspGluLysAsn                              65707580                                                                      GlnMetMetThrThrAsnValTrpValLysGlnGluTrpHisAspTyr                              859095                                                                        LysLeuArgTrpAspProAlaAspTyrGluAsnValThrSerIleArg                              100105110                                                                     IleProSerGluLeuIleTrpArgProAspIleAlaLeuTyrAsnAsn                              115120125                                                                     AlaAspGlyAspPheAlaAlaThrHisLeuThrLysAlaHisLeuPhe                              130135140                                                                     HisAspGlyArgValGlnArgThrProProAlaIleTyrLysSerSer                              145150155160                                                                  CysSerIleAspValThrPhePheProPheAspGlnGlnAsnCysThr                              165170175                                                                     MetLysPheGlySerTrpThrTyrAspLysAlaLysIleAspLeuVal                              180185190                                                                     AsnMetHisSerArgValAspGlnLeuAspPheTrpGluSerGlyGlu                              195200205                                                                     TrpLeuIleAlaAspAlaXaaGlyThrTyrAsnThrArgLysTyrGlu                              210215220                                                                     CysCysAlaGluIleTyrProAspIleThrTyrAlaTyrAlaIleArg                              225230235240                                                                  ArgLeuProLeuPheCysThrIleAsnLeuIleIleProTrpLeuLeu                              245250255                                                                     IleSerCysLeuThrAlaLeuValPheTyrLeuProSerGluCysGly                              260265270                                                                     GluLysIleThrLeuCysIleSerAlaLeuLeuSerLeuThrGlyPhe                              275280285                                                                     LeuLeuLeuIleThrGluIleIleProProThrSerLeuValIlePro                              290295300                                                                     LeuIleGlyGluTyrLeuLeuPheThrMetIlePheValThrLeuSer                              305310315320                                                                  IleAlaIleThrValPheValLeuAsnValHisHisArgSerProArg                              325330335                                                                     ThrHisThrMetProThrTrpValArgSerValPheLeuAspIleVal                              340345350                                                                     ProArgLeuLeuLeuMetLysArgProSerValValLysAspAsnCys                              355360365                                                                     ArgArgLeuIleGluSerMetHisLysMetAlaSerAlaProArgPhe                              370375380                                                                     TrpProGluProGluGlyGluProProAlaThrSerGlyThrGlnSer                              385390395400                                                                  LeuHisProProSerProProPheCysValProLeuAspValProAla                              405410415                                                                     GluProGlyProSerCysLysSerProSerAspGlnLeuProProGln                              420425430                                                                     LysProLeuGluAlaGluLysAspSerProHisProSerProGlyPro                              435440445                                                                     CysArgProProHisGlyThrGlnAlaProGlyLeuAlaLysAlaArg                              450455460                                                                     SerLeuSerValGlnHisMetSerSerProGlyGluAlaValGluGly                              465470475480                                                                  GlyValArgCysArgSerArgSerIleGlnTyrCysValProArgAsp                              485490495                                                                     AspAlaAlaProGluAlaAspGlyGlnAlaXaaGlyAlaLeuAlaSer                              500505510                                                                     ArgAsnSerHisSerAlaGluLeuProProProAspGlnProSerPro                              515520525                                                                     CysLysCysThrCysLysLysGluProSerSerValSerProSerAla                              530535540                                                                     XaaValLysThrArgSerThrLysAlaProProProHisLeuProLeu                              545550555560                                                                  SerProAlaLeuSerArgAlaValGluGlyValGlnTyrIleAlaAsp                              565570575                                                                     HisLeuLysAlaGluAspThrAspPheSerValLysGluAspTrpLys                              580585590                                                                     TyrValAlaMetValIleAspArgIlePheLeuTrpMetPheIleIle                              595600605                                                                     ValCysLeuLeuGlyThrValGlyLeuPheLeuProProTrpLeuAla                              610615620                                                                     GlyMetIle                                                                     625                                                                           (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1876 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: both                                                            (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 73..1581                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GGCCGCAGGCGCAGGCCCGGGCGACAGCCGAGACGTGGAGCGCGCCGGCTCGCTGCAGCT60                CCGGGACTCAACATGCGCTGCTCGCCGGGAGGCGTCTGGCTGGCGCTGGCCGCGTCGCTC120               CTGCACGTGTCCCTGCAAGGCGAGTTCCAGAGGAAGCTTTACAAGGAGCTGGTCAAGAAC180               TACAATCCCTTGGAGAGGCCCGTGGCCAATGACTCGCAACCACTCACCGTCTACTTCTCC240               CTGAGCCTCCTGCAGATCATGGACGTGGATGAGAAGAACCAAGTTTTAACCACCAACATT300               TGGCTGCAAATGTCTTGGACAGATCACTATTTACAGTGGAATGTGTCAGAATATCCAGGG360               GTGAAGACTGTTCGTTTCCCAGATGGCCAGATTTGGAAACCAGACATTCTTCTCTATAAC420               AGTGCTGATGAGCGCTTTGACGCCACATTCCACACTAACGTGTTGGTGAATTCTTCTGGG480               CATTGCCAGTACCTGCCTCCAGGCATATTCAAGAGTTCCTGCTACATCGATGTACGCTGG540               TTTCCCTTTGATGTGCAGCACTGCAAACTGAAGTTTGGGTCCTGGTCTTACGGAGGCTGG600               TCCTTGGATCTGCAGATGCAGGAGGCAGATATCAGTGGCTATATCCCCAATGGAGAATGG660               GACCTAGTGGGAATCCCCGGCAAGAGGAGTGAAAGGTTCTATGAGTGCTGCAAAGAGCCC720               TACCCCGATGTCACCTTCACAGTGACCATGCGCCGCAGGACGCTCTACTATGGCCTCAAC780               CTGCTGATCCCCTGTGTGCTCATCTCCGCCCTCGCCCTGCTGGTGTTCCTGCTTCCTGCA840               GATTCCGGGGAGAAGATTTCCCTGGGGATAACAGTCTTACTCTCTCTTACCGTCTTCATG900               CTGCTCGTGGCTGAGATCATGCCCGCAACATCCGATTCGGTACCATTGATAGCCCAGTAC960               TTCGCCAGCACCATGATCATCGTGGGCCTCTCGGTGGTGGTGACGGTGATCGTGCTGCAG1020              TACCACCACCACGACCCCGACGGGGGCAAGATGCCCAAGTGGACCAGAGTCATCCTTCTG1080              AACTGGTGCGCGTGGTTCCTSCGAATGAAGAGGCCCGGGGAGGACAAGGTGCGCCCGGCC1140              TGCCAGCACAAGCAGCGGCGCTGCAGCCTGGCCAGTGTGGAGATGAGCGCCGTGGCGCCG1200              CCGCCCGCCAGCAACGGGAACCTGCTGTACATCGGCTTCCGCGGCCTGGACGGCGTGCAC1260              TGTGTCCCGACCCCCGACTCTGGGGTAGTGTGTGGCCGCATGGCCTGCTCCCCCACGCAC1320              GATGAGCACCTCCTGCACGGCGGGCAACCCCCCGAGGGGGACCCGGACTTGGCCAAGATC1380              CTGGAGGAGGTCCGCTACATTGCCAATCGCTTCCGCTGCCAGGACGAAAGCGAGGCGGTC1440              TGCAGCGAGTGGAAGTTCGCCGCCTGTGTGGTGGACCGCCTGTGCCTCATGGCCTTCTCG1500              GTCTTCACCATCATCTGCACCATCGGCATCCTGATGTCGGCTCCCAACTTCGTGGAGGCC1560              GTGTCCAAAGACTTTGCGTAACCACGCCTGGTTCTGTACATGTGGAAAACTCACAGATGG1620              GCAAGGCCTTTGGCTTGGCGAGATTTGGGGGTGCTAATCCAGGACAGCATTACACGCCAC1680              AACTCCAGTGTTCCCTTCTGGCTGTCAGTCGTGTTGCTTACGGTTTCTTTGTTACTTTAG1740              GTAGTAGAATCTCAGCACTTTGTTTCATATTCTCAGATGGGCTGATAGATATCCTTGGCA1800              CATCCGTACCATCGGTCAGCAGGGCCACTGAGTAGTCATTTTGCCCATTAGCCCACTGCC1860              TGGAAAGCCCTTCGGA1876                                                          (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 502 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       MetArgCysSerProGlyGlyValTrpLeuAlaLeuAlaAlaSerLeu                              151015                                                                        LeuHisValSerLeuGlnGlyGluPheGlnArgLysLeuTyrLysGlu                              202530                                                                        LeuValLysAsnTyrAsnProLeuGluArgProValAlaAsnAspSer                              354045                                                                        GlnProLeuThrValTyrPheSerLeuSerLeuLeuGlnIleMetAsp                              505560                                                                        ValAspGluLysAsnGlnValLeuThrThrAsnIleTrpLeuGlnMet                              65707580                                                                      SerTrpThrAspHisTyrLeuGlnTrpAsnValSerGluTyrProGly                              859095                                                                        ValLysThrValArgPheProAspGlyGlnIleTrpLysProAspIle                              100105110                                                                     LeuLeuTyrAsnSerAlaAspGluArgPheAspAlaThrPheHisThr                              115120125                                                                     AsnValLeuValAsnSerSerGlyHisCysGlnTyrLeuProProGly                              130135140                                                                     IlePheLysSerSerCysTyrIleAspValArgTrpPheProPheAsp                              145150155160                                                                  ValGlnHisCysLysLeuLysPheGlySerTrpSerTyrGlyGlyTrp                              165170175                                                                     SerLeuAspLeuGlnMetGlnGluAlaAspIleSerGlyTyrIlePro                              180185190                                                                     AsnGlyGluTrpAspLeuValGlyIleProGlyLysArgSerGluArg                              195200205                                                                     PheTyrGluCysCysLysGluProTyrProAspValThrPheThrVal                              210215220                                                                     ThrMetArgArgArgThrLeuTyrTyrGlyLeuAsnLeuLeuIlePro                              225230235240                                                                  CysValLeuIleSerAlaLeuAlaLeuLeuValPheLeuLeuProAla                              245250255                                                                     AspSerGlyGluLysIleSerLeuGlyIleThrValLeuLeuSerLeu                              260265270                                                                     ThrValPheMetLeuLeuValAlaGluIleMetProAlaThrSerAsp                              275280285                                                                     SerValProLeuIleAlaGlnTyrPheAlaSerThrMetIleIleVal                              290295300                                                                     GlyLeuSerValValValThrValIleValLeuGlnTyrHisHisHis                              305310315320                                                                  AspProAspGlyGlyLysMetProLysTrpThrArgValIleLeuLeu                              325330335                                                                     AsnTrpCysAlaTrpPheLeuArgMetLysArgProGlyGluAspLys                              340345350                                                                     ValArgProAlaCysGlnHisLysGlnArgArgCysSerLeuAlaSer                              355360365                                                                     ValGluMetSerAlaValAlaProProProAlaSerAsnGlyAsnLeu                              370375380                                                                     LeuTyrIleGlyPheArgGlyLeuAspGlyValHisCysValProThr                              385390395400                                                                  ProAspSerGlyValValCysGlyArgMetAlaCysSerProThrHis                              405410415                                                                     AspGluHisLeuLeuHisGlyGlyGlnProProGluGlyAspProAsp                              420425430                                                                     LeuAlaLysIleLeuGluGluValArgTyrIleAlaAsnArgPheArg                              435440445                                                                     CysGlnAspGluSerGluAlaValCysSerGluTrpLysPheAlaAla                              450455460                                                                     CysValValAspArgLeuCysLeuMetAlaPheSerValPheThrIle                              465470475480                                                                  IleCysThrIleGlyIleLeuMetSerAlaProAsnPheValGluAla                              485490495                                                                     ValSerLysAspPheAla                                                            500                                                                           (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2450 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: both                                                            (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 267..1775                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GGCTCCTCCCCCTCACCGTCCCAATTGTATTCCCTGGAAGAGCAGCCGGAAAAGCCTCCG60                CCTGCTCATACCAGGATAGGCAAGAAGCTGGTTTCTCCTCGCAGCCAACTCCCTGAGACC120               CAGGAACCACCGCGGCGGCCGGCACCACCTGGACCCAGCTCCAGGCGGGCGCGGCTTCAG180               CACCACGGACAGCGCCCCACCCGCGGCCCTCCCCCCGGCGGCGCGCTCCAGCCGGTGTAG240               GCGAGGCAGCGAGCTATGCCCGCGGCATGGCCCGGCGCTGCGGCCCCGTGGCG293                      MetAlaArgArgCysGlyProValAla                                                   15                                                                            CTGCTCCTTGGCTTCGGCCTCCTCCGGCTGTGCTCAGGGGTGTGGGGT341                           LeuLeuLeuGlyPheGlyLeuLeuArgLeuCysSerGlyValTrpGly                              10152025                                                                      ACGGATACAGAGGAGCGGCTGGTGGAGCATCTCCTGGATCCTTCCCGC389                           ThrAspThrGluGluArgLeuValGluHisLeuLeuAspProSerArg                              303540                                                                        TACAACAAGCTTATCCGCCCAGCCACCAATGGCTCTGAGCTGGTGACA437                           TyrAsnLysLeuIleArgProAlaThrAsnGlySerGluLeuValThr                              455055                                                                        GTACAGCTTATGGTGTCACTGGCCCAGCTCATCAGTGTGCATGAGCGG485                           ValGlnLeuMetValSerLeuAlaGlnLeuIleSerValHisGluArg                              606570                                                                        GAGCAGATCATGACCACCAATGTCTGGCTGACCCAGGAGTGGGAAGAT533                           GluGlnIleMetThrThrAsnValTrpLeuThrGlnGluTrpGluAsp                              758085                                                                        TATCGCCTCACCTGGAAGCCTGAAGAGTTTGACAACATGAAGAAAGTT581                           TyrArgLeuThrTrpLysProGluGluPheAspAsnMetLysLysVal                              9095100105                                                                    CGGCTCCCTTCCAAACACATCTGGCTCCCAGATGTGGTCCTGTACAAC629                           ArgLeuProSerLysHisIleTrpLeuProAspValValLeuTyrAsn                              110115120                                                                     AATGCTGACGGCATGTACGAGGTGTCCTTCTATTCCAATGCCGTGGTC677                           AsnAlaAspGlyMetTyrGluValSerPheTyrSerAsnAlaValVal                              125130135                                                                     TCCTATGATGGCAGCATCTTCTGGCTGCCGCCTGCCATCTACAAGAGC725                           SerTyrAspGlySerIlePheTrpLeuProProAlaIleTyrLysSer                              140145150                                                                     GCATGCAAGATTGAAGTAAAGCACTTCCCATTTGACCAGCAGAACTGC773                           AlaCysLysIleGluValLysHisPheProPheAspGlnGlnAsnCys                              155160165                                                                     ACCATGAAGTTCCGTTCGTGGACCTACGACCGCACAGAGATCGACTTG821                           ThrMetLysPheArgSerTrpThrTyrAspArgThrGluIleAspLeu                              170175180185                                                                  GTGCTGAAGAGTGAGGTGGCCAGCCTGGACGACTTCACACCTAGTGGT869                           ValLeuLysSerGluValAlaSerLeuAspAspPheThrProSerGly                              190195200                                                                     GAGTGGGACATCGTGGCGCTGCCGGGCCGGCGCAACGAGAACCCCGAC917                           GluTrpAspIleValAlaLeuProGlyArgArgAsnGluAsnProAsp                              205210215                                                                     GACTCTACGTACGTGGACATCACGTATGACTTCATCATTCGCCGCAAG965                           AspSerThrTyrValAspIleThrTyrAspPheIleIleArgArgLys                              220225230                                                                     CCGCTCTTCTACACCATCAACCTCATCATCCCCTGTGTGCTCATCACC1013                          ProLeuPheTyrThrIleAsnLeuIleIleProCysValLeuIleThr                              235240245                                                                     TCGCTAGCCATCCTTGTCTTCTACCTGCCATCCGACTGTGGCGAGAAG1061                          SerLeuAlaIleLeuValPheTyrLeuProSerAspCysGlyGluLys                              250255260265                                                                  ATGACGTTGTGCATCTCAGTGCTGCTGGCGCTCACGGTCTTCCTGCTG1109                          MetThrLeuCysIleSerValLeuLeuAlaLeuThrValPheLeuLeu                              270275280                                                                     CTCATCTCCAAGATCGTGCCTCCCACCTCCCTCGACGTGCCGCTCGTC1157                          LeuIleSerLysIleValProProThrSerLeuAspValProLeuVal                              285290295                                                                     GGCAAGTACCTCATGTTCACCATGGTGCTTGTCACCTTCTCCATCGTC1205                          GlyLysTyrLeuMetPheThrMetValLeuValThrPheSerIleVal                              300305310                                                                     ACCAGCGTGTGCGTGCTCAACGTGCACCACCGCTCGCCCACCACGCAC1253                          ThrSerValCysValLeuAsnValHisHisArgSerProThrThrHis                              315320325                                                                     ACCATGGCGCCCTGGGTGAAGGTCGTCTTCCTGGAGAAGCTGCCCGCG1301                          ThrMetAlaProTrpValLysValValPheLeuGluLysLeuProAla                              330335340345                                                                  CTGCTCTTCATGCAGCAGCCACGCCATCATTGCGCCCGTCAGCGCCTG1349                          LeuLeuPheMetGlnGlnProArgHisHisCysAlaArgGlnArgLeu                              350355360                                                                     CGCCTGCGGCGACGCCAGCGTGAGCGCGAGGGCGCTGGAGCCCTCTTC1397                          ArgLeuArgArgArgGlnArgGluArgGluGlyAlaGlyAlaLeuPhe                              365370375                                                                     TTCCGCGAAGCCCCAGGGGCCGACTCCTGCACGTGCTTCGTCAACCGC1445                          PheArgGluAlaProGlyAlaAspSerCysThrCysPheValAsnArg                              380385390                                                                     GCGTCGGTGCAGGGGTTGGCCGGGGCCTTCGGGGCTGAGCCTGCACCA1493                          AlaSerValGlnGlyLeuAlaGlyAlaPheGlyAlaGluProAlaPro                              395400405                                                                     GTGGCGGGCCCCGGGCGCTCAGGGGAGCCGTGTGGCTGTGGCCTCCGG1541                          ValAlaGlyProGlyArgSerGlyGluProCysGlyCysGlyLeuArg                              410415420425                                                                  GAGGCGGTGGACGGCGTGCGCTTCATCGCAGACCACATGCGGAGCGAG1589                          GluAlaValAspGlyValArgPheIleAlaAspHisMetArgSerGlu                              430435440                                                                     GACGATGACCAGAGCGTGAGTGAGGACTGGAAGTACGTCGCCATGGTG1637                          AspAspAspGlnSerValSerGluAspTrpLysTyrValAlaMetVal                              445450455                                                                     ATCGACCGCCTCTTCCTCTGGATCTTTGTCTTTGTCTGTGTCTTTGGC1685                          IleAspArgLeuPheLeuTrpIlePheValPheValCysValPheGly                              460465470                                                                     ACCATCGGCATGTTCCTGCAGCCTCTCTTCCAGAACTACACCACCACC1733                          ThrIleGlyMetPheLeuGlnProLeuPheGlnAsnTyrThrThrThr                              475480485                                                                     ACCTTCCTCCACTCAGACCACTCAGCCCCCAGCTCCAAGTGAGGCCCTT1782                         ThrPheLeuHisSerAspHisSerAlaProSerSerLys                                       490495500                                                                     CCTCATCTCCATGCTCTTTCACCCTGCCACCCTCTGCTGCACAGTAGTGTTGGGTGGAGG1842              ATGGACGAGTGAGCTACCAGGAAGAGGGGCGCTGCCCCCACAGATCCATCCTTTTGCTTC1902              ATCTGGAGTCCCTCCTCCCCCACGCCTCCATCCACACACAGCAGCTCCAACCTGGAGGCT1962              GGACCAACTGCTTTGTTTTGGCTGCTCTCCATCTCTTGTACCAGCCCAGGCAATAGTGTT2022              GAGGAGGGGAGCAAGGCTGCTAAGTGGAAGACAGAGATGGCAGAGCCATCCACCCTGAGG2082              AGTGACGGGCAAGGGGCCAGGAAGGGGACAGGATTGTCTGCTGCCTCCAAGTCATGGGAG2142              AAGAGGGGTATAGGACAAGGGGTGGAAGGGCAGGAGCTCACACCGCACCGGGCTGGCCTG2202              ACACAATGGTAGCTCTGAAGGGAGGGGAAGAGAGAGGCCTGGGTGTGACCTGACACCTGC2262              CGCTGCTTGAGTGGACAGCAGCTGGACTGGGTGGGCCCCACAGTGGTCAGCGATTCCTGC2322              CAAGTAGGGTTTAGCCGGGCCCCATGGTCACAGACCCCTGGGGGAGGCTTCCAGCTCAGT2382              CCCACAGCCCCTTGCTTCTAAGGGATCCAGAGACCTGCTCCAGATCCTCTTTCCCCACTG2442              AAGAATTC2450                                                                  (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 502 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      MetAlaArgArgCysGlyProValAlaLeuLeuLeuGlyPheGlyLeu                              151015                                                                        LeuArgLeuCysSerGlyValTrpGlyThrAspThrGluGluArgLeu                              202530                                                                        ValGluHisLeuLeuAspProSerArgTyrAsnLysLeuIleArgPro                              354045                                                                        AlaThrAsnGlySerGluLeuValThrValGlnLeuMetValSerLeu                              505560                                                                        AlaGlnLeuIleSerValHisGluArgGluGlnIleMetThrThrAsn                              65707580                                                                      ValTrpLeuThrGlnGluTrpGluAspTyrArgLeuThrTrpLysPro                              859095                                                                        GluGluPheAspAsnMetLysLysValArgLeuProSerLysHisIle                              100105110                                                                     TrpLeuProAspValValLeuTyrAsnAsnAlaAspGlyMetTyrGlu                              115120125                                                                     ValSerPheTyrSerAsnAlaValValSerTyrAspGlySerIlePhe                              130135140                                                                     TrpLeuProProAlaIleTyrLysSerAlaCysLysIleGluValLys                              145150155160                                                                  HisPheProPheAspGlnGlnAsnCysThrMetLysPheArgSerTrp                              165170175                                                                     ThrTyrAspArgThrGluIleAspLeuValLeuLysSerGluValAla                              180185190                                                                     SerLeuAspAspPheThrProSerGlyGluTrpAspIleValAlaLeu                              195200205                                                                     ProGlyArgArgAsnGluAsnProAspAspSerThrTyrValAspIle                              210215220                                                                     ThrTyrAspPheIleIleArgArgLysProLeuPheTyrThrIleAsn                              225230235240                                                                  LeuIleIleProCysValLeuIleThrSerLeuAlaIleLeuValPhe                              245250255                                                                     TyrLeuProSerAspCysGlyGluLysMetThrLeuCysIleSerVal                              260265270                                                                     LeuLeuAlaLeuThrValPheLeuLeuLeuIleSerLysIleValPro                              275280285                                                                     ProThrSerLeuAspValProLeuValGlyLysTyrLeuMetPheThr                              290295300                                                                     MetValLeuValThrPheSerIleValThrSerValCysValLeuAsn                              305310315320                                                                  ValHisHisArgSerProThrThrHisThrMetAlaProTrpValLys                              325330335                                                                     ValValPheLeuGluLysLeuProAlaLeuLeuPheMetGlnGlnPro                              340345350                                                                     ArgHisHisCysAlaArgGlnArgLeuArgLeuArgArgArgGlnArg                              355360365                                                                     GluArgGluGlyAlaGlyAlaLeuPhePheArgGluAlaProGlyAla                              370375380                                                                     AspSerCysThrCysPheValAsnArgAlaSerValGlnGlyLeuAla                              385390395400                                                                  GlyAlaPheGlyAlaGluProAlaProValAlaGlyProGlyArgSer                              405410415                                                                     GlyGluProCysGlyCysGlyLeuArgGluAlaValAspGlyValArg                              420425430                                                                     PheIleAlaAspHisMetArgSerGluAspAspAspGlnSerValSer                              435440445                                                                     GluAspTrpLysTyrValAlaMetValIleAspArgLeuPheLeuTrp                              450455460                                                                     IlePheValPheValCysValPheGlyThrIleGlyMetPheLeuGln                              465470475480                                                                  ProLeuPheGlnAsnTyrThrThrThrThrPheLeuHisSerAspHis                              485490495                                                                     SerAlaProSerSerLys                                                            500                                                                           (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1915 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: both                                                            (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 87..1583                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      CCGGCGCTCACTCGACCGCGCGGCTCACGGGTGCCCTGTGACCCCACAGCGGAGCTCGCG60                GCGGCTGCCACCCGGCCCCGCCGGCCATGAGGCGCGCGCCTTCCCTGGTCCTTTTCTTCC120               TGGTCGCCCTTTGCGGGCGCGGGAACTGCCGCGTGGCCAATGCGGAGGAAAAGCTGATGG180               ACGACCTTCTGAACAAAACCCGTTACAATAACCTGATCCGCCCAGCCACCAGCTCCTCAC240               AGCTCATCTCCATCAAGCTGCAGCTCTCCCTGGCCCAGCTTATCAGCGTGAATGAGCGAG300               AGCAGATCATGACCACCAATGTCTGGCTGAAACAGGAATGGACTGATTACCGCCTGACCT360               GGAACAGCTCCCGCTACGAGGGTGTGAACATCCTGAGGATCCCTGCAAAGCGCATCTGGT420               TGCCTGACATCGTGCTTTACAACAACGCCGACGGGACCTATGAGGTGTCTGTCTACACCA480               ACTTGATAGTCCGGTCCAACGGCAGCGTCCTGTGGCTGCCCCCTGCCATCTACAAGAGCG540               CCTGCAAGATTGAGGTGAAGTACTTTCCCTTCGACCAGCAGAACTGCACCCTCAAGTTCC600               GCTCCTGGACCTATGACCACACGGAGATAGACATGGTCCTCATGACGCCCACAGCCAGCA660               TGGATGACTTTACTCCCAGTGGTGAGTGGGACATAGTGGCCCTCCCAGGGAGAAGGACAG720               TGAACCCACAAGACCCCAGCTACGTGGACGTGACTTACGACTTCATCATCAAGCGCAAGC780               CTCTGTTCTACACCATCAACCTCATCATCCCCTGCGTGCTCACCACCTTGCTGGCCATCC840               TCGTCTTCTACCTGCCATCCGACTGCGGCGAGAAGATGACACTGTGCATCTCAGTGCTGC900               TGGCACTGACATTCTTCCTGCTGCTCATCTCCAAGATCGTGCCACCCACCTCCCTCGATG960               TGCCTCTCATCGGCAAGTACCTCATGTTCACCATGGTGCTGGTCACCTTCTCCATCGYCA1020              CCAGCGTCTGTGTGCTCAATGTGCACCACCGCTCGCCCAGCACCCACACCATGGCACCCT1080              GGGTCAAGCGCTGCTTCCTGCACAAGCTGCCTACCTTCCTCTTCATGAAGCGCCCTGGCC1140              CCGACAGCAGCCCGGCCAGAGCCTTCCCGCCCAGCAAGTCATGCGTGACCAAGCCCGAGG1200              CCACCGCCACCTCCACCAGCCCCTCCAACTTCTATGGGAACTCCATGTACTTTGTGAACC1260              CCGCCTCTGCAGCTTCCAAGTCTCCAGCCGGCTCTACCCCGGTGGCTATCCCCAGGGATT1320              TCTGGCTGCGGYCCTCTGGGAGGTTCCGACAGGATGTGCAGGAGGCATTAGAAGGTGTCA1380              GCTTCATCGCCCAGCACATGAAGAATGDCGATGAAGACCAGAGTGTCGCTGAGGACTGGA1440              AGAACGTGGCTATGGTGGTGGACCGGCTGTTCCTGTGGGTGTTCATGTTTGTGTGCGTCC1500              TGGGCTCTGTGGGGCTCTTCCTGCCGCCCCTCTTCCAGACCCATGCAGCTTCTGAGGGGC1560              CCTACGCTGCCCAGCGTGACTGAGGGCCCCCTGGGTTGTGGGGTGAGAGGATGTGAGTGG1620              CCGGGTGGGCACTTTGCTGCTTCTTTCTGGGTTGTGGCCGATGAGGCCCTAAGTAAATAT1680              GTGAGCATTGGCCATCAACCCCATCAAACCAGCCACAGCCGTGGAACAGGCAAGGATGGG1740              GGCCTGGCCTGTCCTCTCTGAATGCCTTGGAGGGATCCCAGGAAGCCCCAGTAGGAGGGA1800              GCTTCAGACAGTTCAATTCTGGCCTGTCTTCCTTCCCTGCACCGGGCAATGGGGATAAAG1860              ATGACTTCGTAGCAGCACCTACTATGCTTCAGGCATGGTGCCGGCCTGCCTCTCC1915                   (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 498 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      MetArgArgAlaProSerLeuValLeuPhePheLeuValAlaLeuCys                              151015                                                                        GlyArgGlyAsnCysArgValAlaAsnAlaGluGluLysLeuMetAsp                              202530                                                                        AspLeuLeuAsnLysThrArgTyrAsnAsnLeuIleArgProAlaThr                              354045                                                                        SerSerSerGlnLeuIleSerIleLysLeuGlnLeuSerLeuAlaGln                              505560                                                                        LeuIleSerValAsnGluArgGluGlnIleMetThrThrAsnValTrp                              65707580                                                                      LeuLysGlnGluTrpThrAspTyrArgLeuThrTrpAsnSerSerArg                              859095                                                                        TyrGluGlyValAsnIleLeuArgIleProAlaLysArgIleTrpLeu                              100105110                                                                     ProAspIleValLeuTyrAsnAsnAlaAspGlyThrTyrGluValSer                              115120125                                                                     ValTyrThrAsnLeuIleValArgSerAsnGlySerValLeuTrpLeu                              130135140                                                                     ProProAlaIleTyrLysSerAlaCysLysIleGluValLysTyrPhe                              145150155160                                                                  ProPheAspGlnGlnAsnCysThrLeuLysPheArgSerTrpThrTyr                              165170175                                                                     AspHisThrGluIleAspMetValLeuMetThrProThrAlaSerMet                              180185190                                                                     AspAspPheThrProSerGlyGluTrpAspIleValAlaLeuProGly                              195200205                                                                     ArgArgThrValAsnProGlnAspProSerTyrValAspValThrTyr                              210215220                                                                     AspPheIleIleLysArgLysProLeuPheTyrThrIleAsnLeuIle                              225230235240                                                                  IleProCysValLeuThrThrLeuLeuAlaIleLeuValPheTyrLeu                              245250255                                                                     ProSerAspCysGlyGluLysMetThrLeuCysIleSerValLeuLeu                              260265270                                                                     AlaLeuThrPhePheLeuLeuLeuIleSerLysIleValProProThr                              275280285                                                                     SerLeuAspValProLeuIleGlyLysTyrLeuMetPheThrMetVal                              290295300                                                                     LeuValThrPheSerIleXaaThrSerValCysValLeuAsnValHis                              305310315320                                                                  HisArgSerProSerThrHisThrMetAlaProTrpValLysArgCys                              325330335                                                                     PheLeuHisLysLeuProThrPheLeuPheMetLysArgProGlyPro                              340345350                                                                     AspSerSerProAlaArgAlaPheProProSerLysSerCysValThr                              355360365                                                                     LysProGluAlaThrAlaThrSerThrSerProSerAsnPheTyrGly                              370375380                                                                     AsnSerMetTyrPheValAsnProAlaSerAlaAlaSerLysSerPro                              385390395400                                                                  AlaGlySerThrProValAlaIleProArgAspPheTrpLeuArgXaa                              405410415                                                                     SerGlyArgPheArgGlnAspValGlnGluAlaLeuGluGlyValSer                              420425430                                                                     PheIleAlaGlnHisMetLysAsnXaaAspGluAspGlnSerValAla                              435440445                                                                     GluAspTrpLysAsnValAlaMetValValAspArgLeuPheLeuTrp                              450455460                                                                     ValPheMetPheValCysValLeuGlySerValGlyLeuPheLeuPro                              465470475480                                                                  ProLeuPheGlnThrHisAlaAlaSerGluGlyProTyrAlaAlaGln                              485490495                                                                     ArgAsp                                                                        __________________________________________________________________________

That which is claimed:
 1. An isolated nucleic acid molecule, comprisinga sequence of nucleotides or ribonucleotides that encodes an α₇ subunitof a human neuronal nicotinic acetylcholine receptor, wherein thesequence of nucleotides or ribonucleotides encoding the α₇ subunit isselected from the group consisting of:(a) a sequence of nucleotides orribonucleotides that encodes a human α₇ subunit and comprises the codingportion of the sequence set forth in SEQ.ID.No:7; (b) a sequence ofnucleotides or ribonucleotides that encodes a human α₇ -subunit and thathybridizes under conditions of high stringency to the complement of theentire coding portion of the sequence of nucleotides set forth inSEQ.ID.No:7; (c) a sequence of nucleotides that encodes an α₇ subunit,hybridizes under conditions of high stringency to the complement of thesequence of nucleotides set forth in SEQ.ID.No:7, and, if it is DNA, isfully complementary or, if it is RNA, is identical to mRNA native to ahuman cell; and (d) a sequence of nucleotides or ribonucleotidesdegenerate with the α₇ subunit encoding sequence of (a), (b) or (c). 2.An isolated nucleic acid molecule that encodes an α₇ subunit of a humanneuronal nicotinic acetylcholine receptor, comprising a sequence ofnucleotides or ribonucleotides, wherein the α₇ subunit comprises thesequence of amino acids set forth in SEQ.ID.No:8.
 3. An isolated nucleicacid molecule, comprising a sequence of nucleotides or ribonucleotidesthat encodes an α₇ subunit of a human neuronal nicotinic acetylcholinereceptor, wherein the sequence of nucleotides or ribonucleotideshybridizes under conditions of high stringency to DNA complementary tomRNA that is native to a human cell and that encodes an α₇ subunit. 4.The nucleic acid molecule of claim 3 wherein the sequence of nucleotidesor ribonucleotides hybridizes under high stringency conditions to thecomplement of the entire coding sequence set forth as nucleotides73-1581 in SEQ.ID.No:7.
 5. A nucleic acid molecule that encodes a humannicotinic acetylcholine receptor subunit, comprising a sequence ofnucleotides or ribonucleotides set forth as nucleotides 73-1581 inSEQ.ID.No:7, wherein the sequence of ribonucleotides contains uracil inplace of the thymine for the sequence set forth in SEQ.ID.No:7. 6.Isolated cells, comprising the nucleic acid molecule of claim 1, whereinsaid cells are bacterial cells, mammalian cells or amphibian oocytes,and the nucleic acid molecule is heterologous to the cells.
 7. The cellsof claim 6, further containing a nucleic acid molecule encoding a βsubunit of human neuronal nicotinic acetylcholine receptor.
 8. The cellsof claim 7, wherein said β subunit is selected from β₂ or β₄.
 9. Thecells of claim 7, wherein said β subunit is β₄.
 10. The cells of claim6, wherein:said cells express functional neuronal nicotinicacetylcholine receptors that contain one or more subunits encoded bysaid nucleic acid molecule; and the cells are eukaryotic cells.
 11. Anisolated human neuronal nicotinic acetylcholine receptor, comprising anα₇ subunit encoded by the nucleic acid molecule of claim
 1. 12. Anisolated human neuronal nicotinic acetylcholine receptor subunit encodedby the nucleic acid of claim
 1. 13. The human neuronal nicotinicacetylcholine receptor of claim 11, further comprising at least onehuman neuronal nicotinic acetylcholine receptor beta subunit.
 14. Amethod for identifying functional neuronal nicotinic acetylcholinereceptor subunits and combinations thereof, said method comprising:(a)introducing the nucleic acid molecule of claim 1 and optionally anucleic acid molecule encoding at least one beta subunit of a humanneuronal nicotinic acetylcholine receptor into mammalian or amphibiancells; and (b) assaying for neuronal nicotinic acetylcholine receptoractivity in cells of step (a).
 15. The nucleic acid molecule of claim 1,wherein said nucleic acid molecule is mRNA.
 16. Isolated mammalian andamphibian cells containing the mRNA of claim
 15. 17. The cells of claim16, wherein said cells further contain mRNA encoding a beta subunit of ahuman neuronal nicotinic acetylcholine receptor.
 18. The nucleic acidmolecule of claim 1, comprising the sequence of nucleotides set forth asnucleotides 73-1581 in SEQ.ID.No:7.
 19. The nucleic acid molecule ofclaim 1, comprising the sequence of nucleotides set forth inSEQ.ID.No:7.
 20. The cells of claim 6, wherein the cells expressneuronal nicotinic acetylcholine receptors.
 21. The cells of claim 7,wherein the cells express neuronal nicotinic acetylcholine receptors.22. The nucleic acid molecule of claim 1, wherein the nucleic acidmolecule is isolated from a DNA library.
 23. The mammalian cells andamphibian oocyte cells of claim 17, wherein the cells express functionalneuronal nicotinic acetylcholine receptors that contain one or moresubunits encoded by the nucleic acid.
 24. The nucleic acid molecule ofclaim 1 that is DNA.